Polyamines and Their Role in Human Disease
The role of polyamine catabolism in anti-tumour drug response R.A. Casero, Jr*1 , Y. Wang*, T.M. Stewart*, W. Devereux*, A. Hacker*, Y. Wang*, R. Smith and P.M. Woster† *Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231, U.S.A., and †Wayne State University, Detroit, MI 48202, U.S.A.
Abstract Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N1 -acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H2 O2 by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H2 O2 as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these antitumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 µM of the polyamine oxidase inhibitor N1 ,N4 -bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H2 O2 produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.
Polyamine metabolism as a therapeutic target The metabolism of polyamines has been pursued as a target for anti-neoplastic therapeutic intervention subsequent to the discovery that polyamines were obligatory factors for growth [1]. Initially, much attention was focused on inhibition of the biosynthetic pathway as a means to inhibit tumour growth, and inhibitors to essentially all of the biosynthetic enzymes have been successfully synthesized and tested [2]. Although much was learned about the requirement of polyamines through the development of inhibitors of polyamine biosynthesis, none of the inhibitors have been Key words: polyamine, polyamine oxidase, spermidine/spermine-N1 -acetyltransferase. Abbreviations used: APAO, acetylpolyamine oxidase; PAO, polyamine oxidase; SSAT, spermidine/spermine-N1 -acetyltransferase; PRE, polyamine response element; BENSpm, N1 ,N11 bis(ethyl)norspermine; PMF-1, polyamine-modulated factor-1; Nrf-2, NF-E2-related transcription factor. 1
To whom correspondence should be addressed (e-mail
[email protected]).
successful as single agents in the treatment of cancer. 2-Difluoromethylornithine (‘DFMO’), an irreversible inhibitor of ornithine decarboxylase (‘ODC’), has been among the most successful of the inhibitors and is currently a frontline agent in the treatment of African sleeping sickness [3–5], and is undergoing extensive testing as a chemopreventive agent, particularly in gastrointestinal cancers [6–9]. Recently, there has been an increased interest in the role that polyamine catabolism may play in determining tumour cell response to agents that interfere with or alter polyamine metabolism. This interest is a direct result of the development of several different anti-tumour polyamine analogues. These analogues, typified by N1 ,N11 -bis(ethyl)norspermine (BENSpm) and N1 -ethyl-N11 -[(cyclopropyl)methyl]-4,8diazaundecane (‘CPENSpm’), decrease intracellular polyamines by down-regulating polyamine biosynthesis and, in specific instances, significantly up-regulating polyamine catabolism [10–16]. It is this effect, the up-regulation of C 2003
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polyamine catabolism, that has become a major focal point of our laboratory.
Spermidine/spermine-N1 acetyltransferase (SSAT) We originally demonstrated that the super-induction of SSAT was associated with the phenotype-specific cytotoxic response of human non-small-cell lung cancer cells to bis(ethyl) polyamine analogues [17,18]. We used the BENSpm-induced human lung cancer cell line NCI H157 as a source of SSAT protein, which was purified and partially sequenced, ultimately resulting in the cloning of SSAT [18,19]. Subsequent to our cloning of SSAT it has been demonstrated that the phenotype-specific expression of SSAT is regulated at each step from transcription to posttranslational stabilization of the translated protein [20–28]. Our laboratory has focused on the transcriptional regulation of SSAT.
Figure 1 Model of PMF-1 activation of SSAT transcription in the presence of inducing polyamine analogues (A) The transcription factor Nrf-2 is constitutively bound to the PRE sequence 5 -TATGACTAA-3 in the SSAT promoter. The basal transcriptional complex appears to be capable of driving low levels of transcription. (B) In the presence of polyamines or SSAT-inducing polyamine analogues, PMF-1 complexes with Nrf-2, and potentially the basal transcriptional complex, and drives enhanced transcription of SSAT.
Elements involved in the transcriptional regulation of SSAT To better understand the mechanisms underlying the transcriptional regulation of SSAT, we sought to identify the controlling elements that exist in the promoter region of the SSAT gene and to determine the protein factors that bind to this region leading to increased SSAT expression in response to polyamines and polyamine analogues. Using a combination of sequential deletions, site-directed mutations and reporter construct analysis, a polyamine response element (PRE) was identified [29]. The 9 bp consensus sequence, 5 -TATGACTAA-3 , was then used to probe an expression library produced from BENSpm-induced lung cancer cells. Using this method, the previously described NFE2-related transcription factor (Nrf-2) [30–33] was identified as a protein that was constitutively bound to the PRE of the SSAT gene [29]. Importantly, significant Nrf-2 expression was only observed in the analogue-responsive cell types and not in cells that do not respond to polyamine analogue exposure with increased SSAT expression. Although these data are consistent with Nrf-2 playing a role in the analogue-induced transcription of SSAT, results from electrophoretic mobility shift analysis indicate that, in the responsive cells, Nrf-2 is constitutively bound to the PRE independent of analogue exposure. Consequently, the possibility that an additional protein factor was involved in SSAT transcription could not be overlooked. To test the hypothesis that an additional transcription factor was involved in analogue-induced expression of SSAT, the leucine-zipper domain of Nrf-2 was used as bait in a yeast two-hybrid strategy. This strategy ultimately identified a transcription co-factor designated polyaminemodulated factor-1 (PMF-1) [34]. PMF-1 does not possess a DNA-binding domain, but must first bind to Nrf-2 through a unique leucine-zipper–coiled-coil interaction C 2003
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[35] to modulate SSAT transcription. The expression of PMF-1, similar to that of SSAT, is significantly increased with analogue treatment in analogue-sensitive cells, but not in analogue-insensitive cell lines [35]. However, the rapid increase in SSAT transcription in response to analogue exposure suggests that pre-existing PMF-1 protein may bind to Nrf-2, leading to a near-immediate increase in SSAT transcription. Consequently, the working model of analogueinduced transcription of SSAT that has emerged suggests that, in the presence of the analogue, PMF-1 protein binds to the PRE-bound Nrf-2 and stimulates the transcription of both SSAT and PMF-1 (Figure 1). It is intriguing to consider that PMF-1 may also be involved in the transcriptional regulation of other, yet to be defined, polyamine-responsive genes.
Potential role of SSAT activity in cellular response to the anti-tumour polyamine analogues SSAT is the rate-limiting step in the two-step eukaryotic catabolism of polyamines [36]. A second enzymic step in this pathway is a constitutive, FAD-dependent, acetylpolyamine oxidase (APAO) that has been characterized enzymically. {It should be noted that at the time of writing Wu and McIntire have submitted human and mouse sequences for an APAO to the GenBank database under the accession numbers AF226657 and NM153783, respectively (also see [36a])}. SSAT catalyses the N1 -acetylation of spermidine and spermine, and APAO then metabolizes the acetylated polyamine to H2 O2 , a toxic reactive oxygen species, 3-acetamidopropanal, and spermidine or putrescine,
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Figure 2
PAOh1 gene splice variants The human PAOh1 gene was found to code for at least four splice variants. The longest splice variant, PAOh1, has an open reading frame (ORF) that codes for 555 amino acids. The shortest splice variant, PAOh3, has an open reading frame that only encodes 190 amino acids. Each of these splice variants produced active proteins when analysed in an in vitro wheatgerm transcription/translation system.
depending on the starting substrate. We initially demonstrated that in the BENSpm-sensitive NCI H157 human lung carcinoma cell line a combination of treatment with the polyamine oxidase (PAO) inhibitor MDL 72,527 and BENSpm led to a delay in the onset of BENSpm-induced apoptosis, suggesting that the production of H2 O2 was one of the major factors in determining cellular sensitivity to the polyamine analogues, thus linking the induction of polyamine catabolism and cytotoxicity [37]. Others have subsequently confirmed the link between H2 O2 production by polyamine catabolism and cell death in different tumour cell types [38]. However, at the time these studies were performed it was assumed that there was only one, constitutive, oxidase that acted primarily on the acetylated polyamines in mammalian cells.
PAOh1 At the time of the discovery that PAO activity could play an active role in determining cellular sensitivity to anti-tumour polyamine analogues, no human PAOs had been successfully cloned. Therefore, we sought to identify a human PAO. Our initial strategy was to use the sequence information from the maize PAO [39–42] to attempt to clone a human PAO by reverse transcriptase-PCR. This method was successful and resulted in the first reported clone of a human PAO (PAOh1) [43]. At the time that the original PAOh1 experiments were performed, the acetylated polyamines were not commercially available. However, the oxidase encoded by PAOh1 was found to readily oxidize spermine. Since APAO has also been suggested to use spermine as a substrate, the results of the initial studies did not positively identify PAOh1 as a new enzyme. Recent results from Vujcic et al. [44] are consistent with the hypothesis that PAOh1 does, in fact, represent a previously unidentified enzyme in eukaryotic polyamine
catabolism that utilizes spermine as its preferred substrate. Importantly, PAOh1 was found to be highly inducible by specific anti-tumour polyamine analogues, demonstrating an added level of complexity in the regulation of polyamine homoeostasis. Soon after the initial cloning of PAOh1, it became apparent that the human PAOh1 gene codes for multiple splice variants that were identified from several different tumour and normal cell types (Figure 2). Using an in vitro TnT (linked in vitro transcription/translation) system we have demonstrated that four splice variants from the human PAOh1 gene each code for enzymes with unique biochemical properties that are capable of using multiple substrates, including spermine, spermidine and N1 -acetylspermine [45]. However, the determination of the relevance of the individual splice variants in situ requires additional experimentation since it is possible that the TnT-produced proteins have a different spectrum of substrate specificities when compared with the naturally occurring enzymes.
Regulation of PAOh1 expression Steady-state PAOh1 mRNA increases rapidly in responsive cell types after exposure to the polyamine analogues [43,46]. These results suggest that the initial regulatory step in PAOh1 expression is at the level of transcription. However, formal run-off transcription analysis has not yet been performed to confirm this. Unlike observations associated with the regulation of SSAT expression, most of the regulation of PAOh1 expresion in response to polyamine analogues appears to occur at the level of changes in PAOh1 mRNA, since increases in oxidase activity are generally correlated with similar increases in PAOh1 mRNA [46]. Although considerably more work is necessary to determine the precise molecular mechanisms that regulate C 2003
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PAOh1 expression, the current data are consistent with the hypothesis that analogue exposure of responsive cells results in an increase in PAOh1 transcription, followed by increased oxidase protein production. There is currently no evidence to suggest that the inducing analogues stabilize the PAOh1 protein in a fashion similar to that observed with SSAT [25,28]. This may serve to explain why the greatest induction observed with analogue-induced PAOh1 occurs in the range of 7–10-fold rather than the 1000-fold induction often observed in SSAT activity in response to the same analogue in specific lung and melanoma tumour phenotypes [46]. It should be noted, however, that the 7–10-fold range is probably an underestimate of the actual level of induction, since the basal oxidase activity capable of using spermine as a substrate in the many cell types we have examined is quite high and is not likely to be specific PAOh1 protein. Once effective antibodies to PAOh1 are available, it will be possible to provide a more precise estimate of fold-induction, using antibody-precipitated cell lysate to determine the actual basal level of PAOh1 activity in uninduced cells.
The potential of PAO as a target for anti-neoplastic intervention Similar to observations with SSAT induction, the high induction of PAOh1 activity is not a global event, but occurs in a phenotype-specific manner, and has thus far only been observed to be significantly increased in specific tumour cell lines [46]. It is important to emphasize that one product of PAOh1 activity is H2 O2 , a known cellular toxin. The possibility of selectively inducing PAOh1 in tumour cells, resulting in high intracellular H2 O2 concentrations, presents an intriguing possibility to be explored. Because of this possibility we are in the process of detailed studies to determine the precise mechanisms that regulate PAOh1 expression in tumour and normal cells. In addition, we are performing structure–activity analyses to determine the basic structural requirements necessary for an effective inducer of PAOh1 activity. It is anticipated that, armed with such information, it may very well be possible to selectively target specific tumour phenotypes with agents that induce PAOh1 and kill cancer cells.
Conclusions Polyamine metabolism represents an attractive target for anticancer therapy. Although considerable progress has been made in the production and testing of agents that inhibit polyamine biosynthesis, new attention is being focused on the role of polyamine catabolism in polyamine homoeostasis and drug response. The demonstration that SSAT activity was associated with cellular response to specific anti-tumour polyamine analogues in several important human solid tumour phenotypes including lung, melanoma, prostate and breast cancers, has increased the hope that modulation of polyamine catabolism can be exploited for therapeutic advantage. C 2003
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Until recently, it was assumed that the catabolism of polyamines was essentially under the rate-limiting control of SSAT. However, our recent cloning and characterization of the PAOh1 gene and its splice variants clearly demonstrates that there is a family of oxidases that certainly adds to the level of complexity associated with regulated catabolism of polyamines. By obtaining a more detailed understanding as to how the two pathways regulating polyamine catabolism, the SSAT/APAO pathway and the PAOh1 pathway, are controlled, it may be possible to specifically target tumour cells with agents that lead to the cytotoxic production of H2 O2 in a tumour-selective manner. The progress that has been made thus far in understanding the multiple levels that control SSAT expression in tumour and normal cells, from the cis- and trans-acting factors that control its transcription, and the detailed understanding of how post-translational stabilization of the protein facilitates the super-induction of SSAT activity in some tumour cell types, represent encouraging steps in the journey to exploit this pathway. Similarly, as more is known about the various regulatory steps in the expression of PAOh1, it may be possible to combine the understanding of the regulation of both enzymes, SSAT and PAOh1, to more efficiently and selectively target tumour cells with newly designed anti-tumour polyamine analogues.
References 1 Pegg, A.E. (1988) Cancer Res. 48, 759–774 2 Marton, L.J. and Pegg, A.E. (1995) Annu. Rev. Pharmacol. Toxicol. 35, 55–91 3 Bacchi, C.J. and Yarlett, N. (1993) Acta Trop. 54, 225–236 4 Pepin, J., Khonde, N., Maiso, F., Doua, F., Jaffar, S., Ngampo, S., Mpia, B., Mbulamberi, D. and Kuzoe, F. (2000) Bull. World Health Org. 78, 1284–1295 5 Pepin, J., Milord, F., Guern, C. and Schechter, P.J. (1987) Lancet 2, 1431–1433 6 Meyskens, Jr, F.L. and Gerner, E.W. (1999) Clin. Cancer Res. 5, 945–951 7 Pegg, A.E., Shantz, L.M. and Coleman, C.S. (1995) J. Cell. Biochem. Suppl. 22, 132–138 8 Carbone, P.P., Pirsch, J.D., Thomas, J.P., Douglas, J.A., Verma, A.K., Larson, P.O., Snow, S., Tutsch, K.D. and Pauk, D. (2001) Cancer Epidemiol. Biomarkers Prev. 10, 657–661 9 Gupta, S., Ahmad, N., Marengo, S.R., MacLennan, G.T., Greenberg, N.M. and Mukhtar, H. (2000) Cancer Res. 60, 5125–5133 10 Bergeron, R.J., Neims, A.H., McManis, J.S., Hawthorne, T.R., Vinson, J.R., Bortell, R. and Ingeno, M.J. (1988) J. Med. Chem. 31, 1183–1190 11 Bergeron, R.J., Feng, Y., Weimar, W.R., McManis, J.S., Dimova, H., Porter, C., Raisler, B. and Phanstiel, O. (1997) J. Med. Chem. 40, 1475–1494 12 Bergeron, R.J., McManis, J.S., Liu, C.Z., Feng, Y., Weimar, W.R., Luchetta, G.R., Wu, Q., Ortiz-Ocasio, J., Vinson, J.R., Kramer, D. and Porter, C.W. (1994) J. Med. Chem. 37, 3464–3476. 13 Bernacki, R.J., Bergeron, R.J. and Porter, C.W. (1992) Cancer Res. 52, 2424–2430 14 Casero, R.A. and Woster, P.M. (2001) J. Med. Chem. 44, 1–26 15 Porter, C.W. and Bergeron, R.J. (1988) Adv. Enzyme Regul. 27, 57–79 16 Porter, C.W., Bernacki, R.J., Miller, J. and Bergeron, R.J. (1993) Cancer Res. 53, 581–586 17 Casero, Jr, R.A., Celano, P., Ervin, S.J., Porter, C.W., Bergeron, R.J. and Libby, P.R. (1989) Cancer Res. 49, 3829–3833 18 Casero, Jr, R.A., Celano, P., Ervin, S.J., Wiest, L. and Pegg, A.E. (1990) Biochem. J. 270, 615–620 19 Casero, Jr, R.A., Celano, P., Ervin, S.J., Applegren, N.B., Wiest, L. and Pegg, A.E. (1991) J. Biol. Chem. 266, 810–814 20 Xiao, L. and Casero, Jr, R.A. (1996) Biochem. J. 313, 691–696 21 Fogel-Petrovic, M., Vujcic, S., Brown, P.J., Haddox, M.K. and Porter, C.W. (1996) Biochemistry 35, 14436–14444
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22 Fogel-Petrovic, M., Vujcic, S., Miller, J. and Porter, C.W. (1996) FEBS Lett. 391, 89–94 23 McCloskey, D.E., Coleman, C.S. and Pegg, A.E. (1999) J. Biol. Chem. 274, 6175–6182 24 McCloskey, D.E. and Pegg, A.E. (2000) J. Biol. Chem. 275, 28708– 28714 25 Coleman, C.S., Huang, H. and Pegg, A.E. (1995) Biochemistry 34, 13423–13430 26 Coleman, C.S., Huang, H. and Pegg, A.E. (1996) Biochem. J. 316, 697–701 27 Coleman, C.S. and Pegg, A.E. (1997) J. Biol. Chem. 272, 12164–12169 28 Coleman, C.S. and Pegg, A.E. (2001) Biochem. J. 358, 137–145 29 Wang, Y., Xiao, L., Thiagalingam, A., Nelkin, B.D. and Casero, Jr, R.A. (1998) J. Biol. Chem. 273, 34623–34630 30 Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I. et al. (1997) Biochem. Biophys. Res. Commun. 236, 313–322 31 Kuroha, T., Takahashi, S., Komeno, T., Itoh, K., Nagasawa, T. and Yamamoto, M. (1998) J. Biochem. (Tokyo) 123, 376–379 32 McMahon, M., Itoh, K., Yamamoto, M., Chanas, S.A., Henderson, C.J., McLellan, L.I., Wolf, C.R., Cavin, C. and Hayes, J.D. (2001) Cancer Res. 61, 3299–3307 33 Toki, T., Itoh, J., Kitazawa, J., Arai, K., Hatakeyama, K., Akasaka, J., Igarashi, K., Nomura, N., Yokoyama, M., Yamamoto, M. and Ito, E. (1997) Oncogene 14, 1901–1910 34 Wang, Y., Devereux, W., Stewart, T.M. and Casero, Jr, R.A. (1999) J. Biol. Chem. 274, 22095–22101
35 Wang, Y., Devereux, W., Stewart, T.M. and Casero, Jr, R.A. (2001) Biochem. J. 355, 45–49 36 Casero, Jr, R.A. and Pegg, A.E. (1993) FASEB J. 7, 653–661 36a Vujcic, S., Liang, P., Diegelman, P., Kramer, D.L. and Porter, C.W. (2003) 370, 19–28 37 Ha, H.C., Woster, P.M., Yager, J.D. and Casero, Jr, R.A. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11557–11562 38 Chen, Y., Kramer, D.L., Diegelman, P., Vujcic, S. and Porter, C.W. (2001) Cancer Res. 61, 6437–6444 39 Binda, C., Coda, A., Angelini, R., Federico, R., Ascenzi, P. and Mattevi, A. (1999) Structure Fold. Des. 7, 265–276 40 Binda, C., Coda, A., Angelini, R., Federico, R., Ascenzi, P. and Mattevi, A. (1998) Acta Crystallogr. D. Biol. Crystallogr. 54, 1429–1431 41 Binda, C., Angelini, R., Federico, R., Ascenzi, P. and Mattevi, A. (2001) Biochemistry 40, 2766–2776 42 Tavladoraki, P., Schinina, M.E., Cecconi, F., Di Agostino, S., Manera, F., Rea, G., Mariottini, P., Federico, R. and Angelini, R. (1998) FEBS Lett. 426, 62–66 43 Wang, Y., Devereux, W., Woster, P.M., Stewart, T.M., Hacker, A. and Casero, Jr, R.A. (2001) Cancer Res. 61, 5370–5373 44 Vujcic, S., Diegelman, P., Bacchi, C.J., Kramer, D.L. and Porter, C.W. (2002) Biochem. J. 367, 665–675 45 Murray-Stewart, T., Wang, Y., Devereux, W. and Casero, R.A. (2002) Biochem. J. 368, 673–677 46 Devereux, W., Wang, Y., Murray-Stewart, T., Hacker, A. and Casero, Jr, R.A. (2002) Proc. Am. Assoc. Cancer Res. 43, 963–964 Received 18 November 2002
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