Chapter 2. Review of the literature
- Table of Contents
- 2.1. Polyamines
- 2.2. Ornithine decarboxylase
- 2.3. Antizyme
2.1. Polyamines
2.1.1. Structure and properties
The polyamines, spermidine, spermine and the diamine precursor, putrescine, are positively charged aliphatic amines that have shown in numerous studies to be essential for normal cell growth and differentiation. Putrescine NH2(CH2)4NH2 and spermidine NH2(CH2)4NH(CH2)3NH2 occur in almost all living species, spermine NH2(CH2)3NH- (CH2)4NH(CH2)3NH2 is less common in prokaryotes. Since their primary and secondary amino groups are all protonated at physiological pH, putrescine is divalent, spermidine trivalent and spermine tetravalent organic cation. In the cells polyamines interact electrostatically with negatively charged moieties such as DNA, RNA, proteins and phospholipids. The unique feature of polyamine structure compared to inorganic cations like Mg2+ or Ca2+ is that they have positive charges at defined distances and between them methylene groups that can participate in hydrophobic interactions. Thus polyamines form stronger and more specific interactions than inorganic cations. (for reviews, see Davis et al. 1992, Marton & Pegg 1995, Thomas & Thomas 2001).
The importance of polyamines for normal cell growth and functions is emphasized by the complex regulative circuitry of synthesis, degradation, uptake and efflux used to adjust and maintain cellular polyamine concentration at a certain level. When cells are deprived of polyamines they cease to grow and proliferate, but usually they do not die (Balasundaram et al. 1991, Balasundaram et al. 1993). Trophic stimuli increases intracellular polyamine concentrations that are highest in rapidly proliferating cells. Generally spermidine and spermine are present in millimolar concentrations, whereas putrescine levels are slightly lower (Morgan 1990, Watanabe et al. 1991, Igarashi & Kashiwagi 2000). However, most polyamines within cells are bound to nucleic acids and other negatively charged structures. Hence, their free and potentially “reactive” concentration is much lower than total concentration.
2.1.2. Physiological roles
2.1.2.1. DNA binding
The most important single characteristics of polyamines is likely to be their ability to bind nucleic acids and especially DNA. Polyamines neutralize the charges on the phosphate groups of DNA, interact with nucleic acid bases and dock into the major or minor grooves of the double helix (Feuerstein et al. 1990, Feuerstein et al. 1991, Tippin & Sundaralingam 1997, Deng et al. 2000). They can increase the melting temperature Tm of DNA in a concentration-dependent manner (Thomas & Bloomfield 1984). The increases are of the order of 10 – 20 ¢ªC at physiological spermine concentration suggesting that polyamines may have a significant role in stabilizing the DNA structure in vivo. Another consequence of polyamine binding to DNA is the condensation of DNA occuring both with naked DNA (Gosule & Schellman 1976, Pelta et al. 1995) and chromatin (Marquet et al. 1986, Sen & Crothers 1986). Immunocytochemical studies of spermidine and spermine have showed that these polyamines are associated with highly compacted mitotic chromosomes (Hougaard et al. 1987, Sauve et al. 1999) although they may have more stabilizing than regulating effect on the chromatin structure during the cell cycle (Laitinen et al. 1998).
Polyamines have also an ability to induce conformational changes to DNA. Their binding has been reported to promote the conversion of the right-handed B-DNA to a lefthanded Z-DNA (Thomas et al. 1991, Bancroft et al. 1994, Hasan et al. 1995) or to an alternative form of right-handed helix, A-DNA (Jain et al. 1989). However, the polyamines can also bind B-DNA so that no change in the secondary structure is detected by Raman spectroscopy (Deng et al. 2000). Z-DNA is induced mainly in blocks of alternating purine-pyrimidine sequences and plays a role in transcriptional control (Herbert & Rich 1999). The other way how polyamines have been suggested to regulate transcription is by inducing bending of DNA after binding to the major groove (Feuerstein et al. 1986, Feuerstein et al. 1989, Rouzina & Bloomfield 1998). Polyamines promote DNA bending by neutralizing the negative charges on DNA phosphate, reducing the energy requirement for bending, and thus facilitating enchanced protein-DNA interactions. DNA bending itself is a major pathway for transcriptional regulation of gene expression (Kerppola 1998, Coulombe 1999). Many DNA binding proteins exert their action by their ability to bend DNA and, in higher organism, cooperative bending produced by multiple transcription factors produce the required response in transcription. Polyamine concentration has been shown to effect the binding of several transcription factors to DNA (Thomas & Thomas 1993, Panagiotidis et al. 1995, Desiderio et al. 1999). In the case of the estrogen receptor, polyamines have been demonstrated to effect directly the conformation of the estrogen responsive element in DNA (Thomas et al. 1997a, Lewis et al. 2000). Polyamines appear also to be involved in signaling pathways that regulate synthesis of a transcription factors or modulate their binding activity via phosphorylation (Wang et al. 1993, Wang et al. 1999, Pfeffer et al. 2000, Wang et al. 2001b).
2.1.2.2. DNA and protein synthesis
When cells suffer from the deprivation of polyamines the first detectable effects are observed in DNA and protein synthesis. DNA replication can be impaired within one cell cycle after seeding cells in the presence of polyamine biosynthesis inhibitors (Fredlund & Oredsson 1996a, Fredlund & Oredsson 1996b) or culturing cells deficient in polyamine biosynthesis without exogenous polyamines (Laitinen et al. 1998). Nuclei isolated from the polyamine depleted cells are reported to synthesize 70-80 % less DNA than nuclei from control cells (Koza & Herbst 1992). These cells may accumulate short DNA fragments corresponding in size to Okazaki fragments (Pohjanpelto & Hölttä 1996). Chromatin from the cells suffering from the prolonged depletion of polyamines is more sensitive to digestion by nucleases (Snyder 1989, Basu et al. 1992, Laitinen et al. 1998). In protein synthesis the effect of polyamine deficiency is seen as an impairement of polysome formation (Hölttä & Hovi 1985), that could be a result of decrease in the initiation and/or elongation of protein synthesis (Takemoto et al. 1983). These effects may be explained by the ability of polyamines to bind and influence secondary structures of mRNA, tRNA, and rRNA (Kusama-Eguchi et al. 1991, Yoshida et al. 1999, Igarashi & Kashiwagi 2000).
2.1.2.3. Cell growth
Depletion of polyamines leads to changes in the expression of numerous growthrelated genes. However, the molecular mechanisms by which these gene expression patters are regulated are mostly unknown and the regulatory pathways involved are just at the beginning of being revealed. Cell cycle arrest after polyamine depletion appears to be mediated by induction of cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1 via p53-dependent mechanisms (Kramer et al. 1999, Ray et al. 1999, Ray et al. 2001), although also p53-independent pathway may exists (Nemoto et al. 2001, Ray et al. 2001). Polyamine depletion has been reported to stabilize both p53 protein and mRNA (Li et al. 2001b). In the cells transformed by the overexpression of polyamine biosynthetic enzymes, p27KIP1 expression is greatly decreased confirming the significance of p27KIP1 for the polyamine responsive cell cycle control (Ravanko et al. 2000). In addition to p21WAF1/CIP1, p27KIP1 and p53, polyamine depletion has effect on the expression of various cyclins. Changes in cyclin A, B1 and D1 expression have been reported (Thomas & Thomas 1994, Thomas et al. 1997b, Marty et al. 2000); with the changes in D1 being most apparent. Above listed alterations in gene expression may be mediated by transcription factor NF-κ B that is rapidly activated in the cells depleted from polyamines (Pfeffer et al. 2001).
2.1.2.4. Apoptosis
Programmed cell death, apoptosis, and cell growth are two sides of the coin; pathways regulating them are partially overlapping and a signal inducing cell proliferation in some cell type or physiological state, can in other cells or circumstances lead to apoptotic death. Thus, it is not surprising that in addition to cell growth, polyamines are involved in the regulation of apoptosis. Paradoxically, it appears that they can act as promoting, modulating or protective agents in apoptosis. (For a review, see Schipper et al. 2000) It has been recognised for years that under certain conditions polyamines have toxic effects on cells (Allen et al. 1979, Gahl & Pitot 1979). Abnormally high polyamine concentrations are well known to be detrimental to cell growth and able to lead to cell death (He et al. 1993, Poulin et al. 1993) that has been shown in several cases to be apoptotic by nature (Tome et al. 1994, Tobias & Kahana 1995, Poulin et al. 1995b, Xie et al. 1997). The oxidation of spermidine and spermine either by serum amine oxidase or by the intracellular FAD-dependent polyamine oxidase produces hydrogen peroxide and aminoaldehydes that are strong inducers of apoptosis (Parchment & Pierce 1989, Ha et al. 1997). However, polyamines appear to be able to induce apoptosis also without oxidation (Brunton et al. 1991, Mitchell et al. 1992, Alhonen et al. 2000), and it is unclear which mechanism plays a major role in cells.
Various studies with different cell systems have shown that the activity of ornithine decarboxylase is fastly and markedly increased after inducing apoptotic cell death (e.g. Donato et al. 1991, Grassilli et al. 1991, Manchester et al. 1993, Desiderio et al. 1995, Penning et al. 1998, Lindsay & Wallace 1999). This leads in some cases, as one could expect, to increases in cellular polyamine levels, but at least equally often the polyamine concentrations are actually decreased. The irreversible inhibitor of ODC, α- difluoromethylornithine (DFMO), inhibits apoptosis in nearly all cell systems where it has been tested (e.g. Piacentini et al. 1991, Manchester et al. 1993, Monti et al. 1998, Penning et al. 1998, Ray et al. 2000). However, it is worth noticing that DFMO treatment up-regulates S-adenosylmethionine decarboxylase (AdoMetDC), another of the key enzymes in polyamine biosynthesis, and although putrescine and spermidine levels are decreased by DFMO, spermine levels are often increased. Interestingly, inhibitors of AdoMetDC (Kaneko et al. 1998, Penning et al. 1998, Satoh et al. 1999, Ray et al. 2000) and spermidine/spermine analogs (McCloskey et al. 1996, Kramer et al. 1997, Shah et al. 2001) generally induce or increase apoptosis. ODC activity effects mostly putrescine levels whereas AdoMetDC provides decarboxylated S-adenosylmethionine for spermidine and spermine synthesis, suggesting that putrescine and higher polyamines could have different roles in the apoptotic cell death. ODC may in some cases have a very active role in the regulation of apoptosis. It has been suggested to be a direct mediator of apoptosis induced by overexpression of c-myc proto-oncogene (Packham & Cleveland 1994). This suggestion was based on the observation that enforced expression of ODC, like c-Myc, was sufficient to induce accelerated cell death following IL-3 withdrawal from murine myeloid cells. In this context, ODC is a mediator of c-Myc-induced apoptosis since c-Myc regulates ODC expression at the level of transcription (Bello- Fernandez et al. 1993).
Although accumulation of polyamine levels appears to be able to trigger apoptosis, decrease in polyamine levels, especially of spermidine and spermine, seems to be a more common feature in apoptosis (e.g. Manchester et al. 1993, Tome et al. 1997, Penning et al. 1998, Moffatt et al. 2000, Nitta et al. 2001). It is highly conceivable that very low levels of polyamines may actually promote apoptosis. Firstly, depletion of polyamines can lead to cell cycle arrest or apoptosis by affecting the p53/p21/p27 cell cycle regulatory pathway (Kramer et al. 1999, Ray et al. 1999, Li et al. 1999b). Secondly, polyamines are important in the regulation of ion transport and the stabilization of important cellular components such as cell membranes and chromatin structures. Therefore depletion of polyamine levels might induce destabilization of important cell structures, leading to loss of cell integrity and finally inducing cell death (Schipper et al. 2000). On the other hand, the depletion of polyamines even after cell-cycle arrest may not be sufficient to induce apoptosis alone (Li et al. 1999b, Li et al. 2001a), but may sensitize cells to apoptosis induced by other factors. The altered susceptibility after polyamine depletion seems to be inducer-specific implicating that polyamines are involved in the regulation of some apoptotic pathways, but not all of them (Stefanelli et al. 2001, Li et al. 2001a). Not surprisingly, spermidine and/or spermine have protective effects against apoptosis in several cell types ranging from neurons (Harada & Sugimoto 1997), Ehrlich ascites tumor (Moffatt et al. 2000) and B cell lymphoma (Nitta et al. 2001) cells to paracite Trypanosoma cruzi (Piacenza et al. 2001). Again the protective effect can be specific to the pathway inducing apoptosis (Hegardt et al. 2000) and may be mediated by activation of transcription of genes required for cell proliferation and survival (Shah et al. 2001).
Although the working mechanisms of polyamines in apoptosis are still inconclusive, it is obvious that excessive levels as well as extremely low levels of polyamines interfere with their specific cellular interactions and effect on their important physiological activities. It has been suggested, that the real physiological significance of polyamine synthesis may reside in cell cycle control and cell survival (Schipper et al. 2000). Therefore, the early induction of ODC often observed during apoptosis may be related to the progression of the cell into a cell cycle phase until a checkpoint is reached from which apoptosis is triggered as a result of cell death-inducing signals. Alternatively, in conditions where apoptosis is inhibited and/or cells have genetic lesions, constitutive ODC expression can lead to cell transformation and deregulated cell growth as observed (Auvinen et al. 1992, Moshier et al. 1993, Auvinen et al. 1997). After the cell cycle check-point polyamine levels may actually decrease during the apoptotic process.
2.1.2.5. Hypusine synthesis
For a long time the only highly specific and unequivocally established function of polyamines was to provide 4-aminobutyl moiety for a synthesis of an unusual amino acid hypusine (Park et al. 1981, Park et al. 1982). The aminobutyl group is transferred from spermidine to a highly conserved lysine of eukaryotic translation initiation factor 5A (eIF- 5A) that is together with a variant form eIF-5A2 the only protein known to contain hypysine (Park et al. 1993, Jenkins et al. 2001). Hypusine is formed in two steps: at first deoxyhypusine synthase (EC 1.1.1.249) catalyzes the transfer of an aminobutyl moiety to a lysine residue to form a deoxyhypusine residue that is subsequently converted to hypusine in a reaction catalyzed by deoxyhypusine hydroxylase (EC 1.14.99.29). Hypusination is required for the biological activity of eIF-5A (Park 1989, Smit-McBride et al. 1989). Hypusine formation is tightly coupled to cell proliferation; its formation may increase by 30-fold after growth-stimulus, and it is essential for cell survival (Chen & Chen 1997, Park et al. 1997). Distruption of either the eIF-5A or deoxyhypusine synthase gene in yeast leads to a lethal phenotype (Schnier et al. 1991, Sasaki et al. 1996, Park et al. 1998). Inhibition of deoxyhypusine synthase in mammalian cells causes growth arrest (Park et al. 1994, Chen et al. 1996, Shi et al. 1996), cell death (Tome & Gerner 1997), or tumor differentiation (Chen et al. 1996). However, hypusine-containing eIF-5A is not required for global protein synthesis. In a conditionally eIF-5A-deficient yeast, the protein synthesis is inhibited only by about 30 % in nonpermissive conditions, and this is accompanied by a slight change in the polysome profile (Kang & Hershey 1994). Rather, activated eIF-5A appears to facilitate the translation of specific subsets of mRNA, maybe the subset of mRNAs required for cell division, and hence the requirement of eIF-5A for cell proliferation would be explained (Park et al. 1997). The significance of eIF-5A for cell proliferation is further emphasized by the facts that the recently found tissue-specific eIF-5A2 variant is expressed, in addition to testis and brain, strongly in a colorectal adenocarcinoma cell line (Jenkins et al. 2001) and that it has been isolated as a candidate oncogene related to the development of ovarian cancer and certain other solid tumors (Guan et al. 2001). Putrescine accumulation in the DFMO resistant cell line after removal of DFMO from the culture induces apoptosis (Tome et al. 1997) that have been attributed to the inhibition of hypusine formation. The generality of this phenomenon in other cell types remains to be determined. Interestingly, in the tomato plant eIF-5A may facilitate translation of the mRNAs required for the programmed cell death (Wang et al. 2001d).
Although it seems clear that hypusine containing eIF-5A is needed for protein synthesis, the way in which it is, is not fully understood. The protein was initially identified as a putative translation initiation factor based on its ability to stimulate methionyl puromycin synthesis under in vitro conditions (Kemper et al. 1976). More recent evidence suggests that eIF-5A facilitates protein synthesis by promoting nuclear transport of specific mRNAs (Ruhl et al. 1993, Katahira et al. 1994). It has also been proposed that eIF-5A may be involved in mRNA turnover, acting downstream of decapping (Zuk & Jacobson 1998). A tentative consensus mRNA sequence for eIF-5A binding has been identified (Xu & Chen 2001). The consensus is present in over 400 human ESTs, but it’s not known whether these represent physiological substrates of eIF- 5A.
2.1.2.6. Other roles
During the last ten years polyamines have been shown to function as endogenous activators and/or blockers of several major classes of cation channels. They are able to activate, inhibite or block NMDA receptors (Ransom & Stec 1988, Williams et al. 1989, Williams 1997) and to block AMPA and kainate receptors (Donevan & Rogawski 1995, Kamboj et al. 1995). All these three receptor groups belong to the class of glutamateactivated receptor channels. Other cation channels blocked by polyamines are inwardly rectifying K+ channels (Fakler et al. 1994, Ficker et al. 1994, Lopatin et al. 1994), nicotinic acetylcholine receptor channels (Haghighi & Cooper 1998, Haghighi & Cooper 2000), cyclic nucleotide-gated channels (Lu & Ding 1999), and voltage-gated Ca2+ (Scott et al. 1993) and Na+ (Huang & Moczydlowski 2001) channels. These observations have raised a lot of interest and the role of polyamines in these contexts is discussed in detail in the chapters 2.1.3 and 2.1.4.
In addition to those discussed above, new functions for polyamines are proposed regularly. One of the most exciting ones is the suggestion that polyamines could modulate the synthesis of nitric oxide (Southan et al. 1994, Szabo et al. 1994, Baydoun & Morgan 1998). This modulation may be mediated at least partly by down-regulation of L-arginine transport (Mössner et al. 2000). Vice versa, nitric oxide has reported to be able to effect polyamine biosynthesis by inhibiting ornithine decarboxylase enzyme (Bauer et al. 2001, Ignarro et al. 2001). Thus, the regulation of these versatile small-molecular modulators of cellular functions may be partially interconnected.
2.1.3. Polyamine metabolism in mammals
2.1.3.1. Biosynthesis
Most living organisms are capable of synthesizing polyamines from the precursor amino acids, arginine and methionine (Davis et al. 1992, Marton & Pegg 1995, Morgan 1999). The metabolic pathways for the synthesis and interconversion of polyamines are well known and established. They are shown in Fig. 1. The first steps committed to polyamine biosynthesis are the decarboxylations of ornithine to form putrescine and Sadenosylmethionine to form decarboxylated S-adenosylmethionine. These irreversible reactions are catalysed by ornithine decarboxylase (ODC, EC 4.1.1.17) and Sadenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50), respectively. Ornithine may be derived from plasma, or intracellular arginine can be converted to ornithine by arginase (EC 3.5.3.1.) in a reaction of the urea cycle pathway. S-adenosylmethionine is a common donor of methyl groups in cells and is formed when an enzyme called ATP:Lmethionine S-adenosyltransferase (EC 2.5.2.6) catalyzes activation of L-methionine. Once AdoMet has been decarboxylated, it is no longer available for transmethylation reactions in the cell.
Polyamine biosynthesis is controlled mainly by two key enzymes, ODC and AdoMetDC. Their activities are rapidly increased or decreased as a response to various positive or negative stimuli. This is based on their fast turnover rate, the half-life of enzyme activity is between 10 and 20 minutes for ODC (Seely et al. 1982a, Isomaa et al. 1983) and from 30 to 60 for AdoMetDC (Pegg 1979, Shirahata & Pegg 1985). Regulation of ODC is discussed later in the chapter 2.2.3. AdoMetDC is regulated at the level of transcription, translation, post-translational processing and protein degradation. Increased levels of mammalian AdoMetDC mRNA, which presumably are due to changes in transcription, have been seen in response to growth promoting factors and to a decline in spermidine produced by a variety of inhibitors (Shirahata & Pegg 1986, Pajunen et al. 1988, Jänne et al. 1991, Svensson et al. 1997). Insulin also increases AdoMetDC mRNA synthesis and an insulin-responsive element has been found in the rat AdoMetDC promotor (Soininen et al. 1996).
In virtually all of the circumstances reported in which enhanced levels of AdoMetDC mRNA have been observed, these increases in mRNA are insufficient to account for the increases in AdoMetDC protein content (see e.g. Pajunen et al. 1988, Persson et al. 1989, Stjernborg et al. 1993, Svensson et al. 1997). All mammalian AdoMetDC mRNAs have a long 5"UTR of about 330 nucleotides, the sequence of which is very highly conserved. The leader sequence contains a small internal open reading frame (ORF) that is located close to 5" terminus (11-14 nucleotides from terminus), is identical among all reported mammalian cDNAs, is in perfect context for translation and has been shown to be translated in vitro (Hill & Morris 1992, Hill & Morris 1993, Raney et al. 2000). ORF codes a peptide of six amino acids with a sequence MAGDIS. High polyamine levels repress the translation of AdoMetDC mRNA efficiently (Kameji & Pegg 1987b) and that repression is not dependent on the secondary structure of 5"UTR of mRNA (Shantz et al. 1994), but requires the presence of ORF in the leader sequence (Ruan et al. 1996). AdoMetDC is synthesized as a proenzyme which then undergoes an internal processing reaction forming α and β subunits and a pyruvate prosthetic group, which is located at the amino terminus of the α subunit (Shirahata & Pegg 1986, Stanley et al. 1989). The processing and activity of AdoMetDC are increased by putrescine, providing a means by which the increased availability of putrescine raises the formation of decarboxylated AdoMet (Kameji & Pegg 1987a, Pegg et al. 1988). The degradation of AdoMetDC is also regulated, but at present, the mechanisms for the degradation and for the alterations in degradation seen in response to polyamines, inhibitors and other stimuli are unknown (Shirahata & Pegg 1985, Autelli et al. 1991, Svensson et al. 1997).
Spermidine and spermine synthases are considered to have only a minor role in the regulation of intracellular polyamine levels. They both are constitutively expressed and stable enzymes regulated mainly by the availability of their substrate, decarboxylated adenosylmethionine. Nevertheless, there are several reports about increased spermidine synthase activity after growth promoting stimuli (Hannonen et al. 1972, Oka et al. 1977, Käpyaho et al. 1980, Korpela et al. 1981, Kauppinen 1995). Increase is dependent on new protein synthesis and could be caused both by increased transcription of spermidine synthase gene and more efficient translation of spermidine synthase mRNA (Kauppinen 1995). Transforming growth factor β 1 that inhibits growth of many cell lines has been suggested to inhibit transcription of the spermidine synthase gene (Nishikawa et al. 1997).
Spermine synthase enzyme is found only in eukaryotes and it is not essential for growth in yeast or mammalian cells (Hamasaki-Katagiri et al. 1998, Korhonen et al. 2001). Gyro (Gy) mice that are deficient in spermine synthase and PHEX gene (regulating phosphate metabolism) show compared to mice deficient only in PHEX gene, reduced viability and fertility, lower body weight and bodily growth, reduced skeletal mineralization and deficiencies in neurological function although analysis of brain tissue revealed no gross or histological abnormalities (Lorenz et al. 1998, Meyer et al. 1998, Mackintosh & Pegg 2000). Fibroblasts taken from these mice were sensitized to ultraviolet irradiation and alkylating agents suggesting that spermine is needed to protect chromosomal DNA (Mackintosh & Pegg 2000, Nilsson et al. 2000b, Stefanelli et al. 2001).
The overruling theme in the regulation of polyamine biosynthesis appears to be to keep polyamine levels within certain limits: to avoid too low and, on the other hand, too high polyamine concentrations. This is well manifested in the results obtained from experiments with transgenic mice. Although overexpression of ODC results in the increase in putrescine levels, only minimal changes have been detected in the concentration of spermidine and spermine (Halmekytö et al. 1991a, Halmekytö et al. 1993, Alhonen et al. 1996). Similarly, transgenic mice overexpressing AdoMetDC (Heljasvaara et al. 1997) or spermidine synthase (Kauppinen et al. 1993) displayed no marked changes in the spermidine or spermine levels, not even after crossbreeding these mice with the mice overexpressing ODC. Indispensability of polyamines has been demostrated also by disruption of the ODC gene in embryonic stem cells and genereating mice harboring a disrupted ODC gene (Pendeville et al. 2001). ODC-heterozygous mice were viable, normal, and fertile, whereas the completely ODC-deficient embryos were capable of uterine implantation on the embryonic day 3.5 and induced maternal decidualization, but failed to develop substantially thereafter.
2.1.3.2. Catabolism and interconversion
In contrast to the extensive studies of polyamine biosynthesis, polyamine degradation has received much less attention untill quite recent years. Mammalian cells have two pathways for polyamine catabolism. An interconversion or recycling pathway converts spermidine and spermine back to putrescine. Terminal catabolic pathway oxidizes polyamines forming aminoaldehydes that cannot be recycled to polyamines. (For reviews, see Casero & Pegg 1993, Seiler 2000)
The interconversion pathway (Fig. 1) is initiated in an acetylation reaction catalyzed by spermidine/spermine N1-acetyltransferase (SSAT, EC 2.3.1.57). The acetyl group is transferred from acetyl-coenzyme A to an aminopropyl moiety of spermine or spermidine. The N1-acetylspermidine or N1-acetylspermine is then oxidized by the constitutive intracellular flavin adenine dinucleotide-dependent polyamine oxidase (PAO, EC 1.4.3.4.), which cleaves the polyamine at a secondary amino nitrogen to release 3- acetamidopropanal and putrescine or spermidine, respectively. Polyamines and their acetylated derivatives are also acted upon at the primary amino groups by diamine oxidases, which are copper-containing amine oxidases (EC 1.4.3.6.). These reactions belong to terminal catabolism of polyamines. Products of reactions include γ - aminobutyric acid, 3-acetamidopropanal, hydrogen peroxide (H2O2) and ammonia. SSAT acetylates the aminopropyl end of spermidine forming N1-acetylspermidine. However, mammalian cells contain also N8-acetylspermidine (Erwin et al. 1984, Pegg et al. 1990). Furthermore, N1,N8-diacetylspermidine (Hiramatsu et al. 1995) and N1,N12- diacetylspermine (van den Berg et al. 1988) have been found in urine. Their functions and enzymes participating in their synthesis are unknown. It has been suggested that acetylation at N8 directs spermidine to be transported from nucleus to cytoplasm (Casero & Pegg 1993).
Tissue polyamine oxidase activity is usually so high that intracellular levels of N1- acetylspermidine or N1-acetylspermine are below the limits of detection, thus the rate of polyamine degradation is regulated by the activity of SSAT. The amount and activity of SSAT are low in most cell types, but are efficiently induced by a number of factors including various toxic agents, hormones, growth factors, polyamines and polyamine analogs (Casero & Pegg 1993, Thomas & Thomas 2001). Polyamines induce the synthesis of polyamine modulated factor 1 (PMF-1) that is a putative cotranscription factor binding to polyamine responsive element in SSAT promotor (Wang et al. 1999, Wang et al. 2001a). Similarly to ODC and AdoMetDC, SSAT has a very short half-life; that of SSAT is about 15 minutes (Matsui & Pegg 1981, Persson & Pegg 1984). Polyamine analogues greatly increase the half-life of SSAT (Parry et al. 1995, Fogel- Petrovic et al. 1996) apparently by inhibiting ubiquitination of the enzyme and preventing its targeting to proteosomal degradation (Coleman & Pegg 2001). Polyamine oxidase was cloned very recently and although it is considered to be constitutively expressed enzyme, its mRNA and activity were induced in non-small cell lung carcinoma cell line by polyamine analog N1, N11-bis(ethyl)norspermine 5- and 3-fold, respectively (Wang et al. 2001c).
Effects of SSAT overexpression have been studied extensively using transgenic animals. Tissues of these mice showed markedly distorted polyamine pools, which in most cases were characterized by the appearance of N1-acetylspermidine, a massive accumulation of putrescine, and significant decreases in spermidine and/or spermine pools (Pietilä et al. 1997, Suppola et al. 1999). The most striking phenotypic change was permanent hair loss likely due to overaccumulation of putrescine (Pietilä et al. 2001). Inducible overexpression of SSAT gene in rat under methallothionein promotor led to acute pancreatitis (Alhonen et al. 2000). In mice the overexpression protected brain from kainate-induced toxicity (Kaasinen et al. 2000), but enhanced general sensitivity to the polyamine analog N1, N11-diethylnorspermine (Alhonen et al. 1999). Interestingly, a hybrid transgenic mice overexpressing both ODC and SSAT under methallothionein promotor showed further accelerated catabolism of hepatic polyamines manifested in a massive putrescine accumulation and in an extreme reduction of spermidine and spermine pools (Suppola et al. 2001). The latter may be partly due to enhanced efflux of polyamines. These results strongly suggest that the catabolisms is the overriding regulatory mechanism in the polyamine metabolism, and that the major aim of the regulation is to prevent an over-accumulation of the higher polyamines.
2.1.3.3. Transport
Most cells have the capacity to synthesise polyamines to a greater or lesser extent and although, historically, it was believed that de novo biosynthesis was by far and away the main supply of polyamines in cells, it is now clear that this is not the case. Additional polyamines are required in the cells with high polyamine demand, such as tumors and normal but rapidly proliferating cells. Exogenous polyamines are also transported to cells when synthesis is disturbed or to organisms that do not produce polyamines. They may be obtained from dietary sources, through synthesis by intestinal microorganisms, or by the release from other cells. (for a review, see Seiler et al. 1996)
All mammalian cells have an active polyamine transport system. Most cells take up polyamines by carrier-mediated and energy-dependent mechanisms, but the nature of the energy coupling is still unclear. Transport is at least partly driven by membrane potential (Poulin et al. 1995a, Poulin et al. 1998). Transport of putrescine and spermidine may be sodium-sensitive, but not sodium-dependent, whereas spermine uptake is not effected by changes in extracellular sodium (Morgan 1999). Divalent cations such as Ca2+, Mg2+ and Mn2+ are essential at least for putrescine and spermidine transport (Brachet et al. 1995, Poulin et al. 1998). The number of carriers involved in the uptake is still debated, and it appears that the number can vary according to cell type. Many cells apparently have a single transporter for putrescine, spermidine and spermine (Seiler et al. 1996). The affinity for the transporter increases with the number of positive charges from putrescine to spermine. However, multiple carrier types per cell are not uncommon. For example, in human umbilical vein endothelial cells there seem to be two carriers, one capable of transporting all three amines and another shared by spermidine and spermine (Morgan 1992). Furthermore, porcine aortic endothelial cells have been reported to posses three carriers, one for both polyamines and one for putrescine (Bogle et al. 1994). Generally the specificity of polyamine transport is not stringent. Derivatives with alkyl substituents on the primary amino groups (Porter et al. 1987) or substituted carbon chains (Sarhan et al. 1987) are transported by different cell lines, as well as biogenic amine agmatine (Satriano et al. 2001). Even compounds with relatively poor structural resemblance to the natural polyamines, such as AdoMetDC inhibitor methylglyoxal bis(guanyl-hydrazone) (Alhonen-Hongisto et al. 1984) and widely used herbicide paraquat (Byers et al. 1987) may share the same transport system with polyamines.
The proteins and composition of polyamine transport system in mammalian cells are not known. Using photoaffinity labelling methods, several polyamine-binding proteins have been detected in the plasma membrane of mammalian cells, but their identification and participation in polyamine transport are still unconfirmed (Felschow et al. 1995, Felschow et al. 1997). In addition to depletion of intracellular polyamine levels, growth stimulus enhances transport (Seiler et al. 1996). As a rule, factors that increase polyamine formation enhance also their uptake from environment. High intracellular polyamine levels inhibit transport from the environment. This effect is at least partly mediated by the antizyme (He et al. 1994, Mitchell et al. 1994), a multifunctional protein that became originally known as an inhibitor of ODC.
Intestinal mucosa and alveolar epithelium are two tissues where polyamine transport appears to be of particular importance. In intestinal mucosa enterocytes take up polyamines from the gut lumena. The transporting capacity of a single enterocyte is not different from that of other cells in the body, but there are an enourmous number of cells in mucosa. In addition, passive diffusion through epithelium without uptake to enterocytes may contribute significantly to the total transport of polyamines (Milovic et al. 2001). Polyamines taken up from intestine have been shown to be important for the regeneration and growth of the mucosa (Osborne & Seidel 1989, Wang et al. 1991) and contribute to the growth of tumors (Sarhan et al. 1989, Sarhan et al. 1992). Alveolar epithelial cells are endowed with a very efficient polyamine uptake system (Hoet & Nemery 2000), but the physiological significance of the polyamine transport to epithelium is still obscure. Polyamine levels have been suggested to be linked to pulmonary hypertension induced by chemicals (Olson et al. 1989) or hypoxia (Atkinson et al. 1987). In a small number of studies it has been observed that polyamines can contribute to the suppression of immunologic reactions in the lung (reviewed in Seiler & Atanassov 1994).
Excretion of polyamines from mammalian cells is much less studied than uptake. Most articles published are about the excretion of polyamines and their acetylated derivatives to urine in healthy and diseased individuals, the individuals being either laboratory animals or human patients (see e.g. Heffner et al. 1995, O"Brien et al. 1995, Hyltander et al. 1998, Langen et al. 2000). At the cellular level excretion is avery poorly understood subject. Polyamine uptake-deficient CHO (Byers et al. 1994) and COS (Hyvönen et al. 1994) cells are able to release polyamines, therefore uptake and export appear to be mediated by different transport systems. Efficient release by diffusion is unlikely in view of the hydrophilic character of polyamines and carrier-mediated export is implied also by studies on excretion from human cancer cell lines (Mackarel & Wallace 1993, Mackarel & Wallace 1994) and erythrocyte cells (Fukumoto & Byus 1996). It has been suggested that antizyme may regulate also excretion (Sakata et al. 2000).
2.1.4. Polyamines in the central nervous system
The essential role of polyamines in the development and differentiation of central nervous system is well documented although their exact role in neurogenesis is not known. They might play an important role in neuronal cell division, differentiation, axonogenesis, synaptogenesis and synaptic plasticity. Likewise, a lot of research effort has been directed to investigate the involvement of polyamines in various pathological conditions of the CNS. Enhanced ODC activity is a common response to different pathological stimuli in the brain: physical, thermal, chemical and metabolic stress all induce increase in ODC activity. (for reviews, see Kauppinen & Alhonen 1995, Johnson 1998, Bernstein & Müller 1999, Seiler 2000).
ODC and AdoMetDC are characteristically regulated in the brain during development. In mammalian brain ODC activity as well as the amount of immunoreactive enzyme protein, are highest around the day of birth (Anderson & Schanberg 1972, Laitinen et al. 1982, Onoue et al. 1988, Morrison et al. 1998). During the few first weeks or months brain ODC activity declines to a low adult level. ODC activity in adult mouse brain is about 70-fold lower than at the time of birth (Suorsa et al. 1992). AdoMetDC activity is very low after birth and increases as the brain matures (Suorsa et al. 1992, Morrison et al. 1993a). This increase is 6-fold in human and 8-fold in mouse brain.
Extracellular polyamines are known to regulate the N-methyl-D-aspartate (NMDA) subtype of glutamate-activated receptor channels in the CNS (Ransom & Stec 1988, Williams et al. 1989), which are believed to have a major physiological functions in the induction of long-term potentiation and in the regulation of embryonal neuronal development (Bliss & Collingridge 1993, Schlaggar et al. 1993). Long-term potentiation is thought to underlie certain types of learning and memory. Transgenic mice overexpressing ODC exhibit a significantly elevated seizure threshold to chemical and electrical stimuli, and impaired performance in spatial learning and memory tests (Halonen et al. 1993). Further analysis in vitro using hippocampal slices revealed that the excitatory synaptic transmission was altered, but no alterations in long-term potentiation were detected, although it is possible that extracellular putrescine levels in slices were not comparable to in vivo situation (Pussinen et al. 1998).
Intracellular polyamines in CNS are responsible for intrinsic gating and rectification of strong inward rectifier K+ (Kir) channels (Fakler et al. 1994, Ficker et al. 1994, Lopatin et al. 1994) and nicotinic acetylcholine receptor ion channels (Haghighi & Cooper 1998, Haghighi & Cooper 2000, Bixel et al. 2001). Kir channels stabilise resting membrane potential in both excitable and non-excitable cells, and control the excitability treshold in neurons and muscle cells (Reimann & Ashcroft 1999, Oliver et al. 2000). The role of polyamines in their regulation is discussed in the chapter 2.1.5. Nicotinic acetylcholine receptor calcium channels are widespread in the nervous system where they function as postsynaptic receptors to excite neurons or as presynaptic receptors to modulate neurotransmitter release (Sargent 1993, Role & Berg 1996). They have been implicated in a wide variety of cognitive functions, including visual and auditory processing, nociception, and attention and memory mechanism (Picciotto et al. 1995, Bannon et al. 1998, Xiang et al. 1998, Marubio et al. 1999, Vetter et al. 1999). The location of polyamine binding sites in a receptor have been mapped (Bixel et al. 2001), but otherwise little is known about the mechanism and the physiological significance of rectification exerted by polyamines.
Increase in ODC activity is a common response to different pathological stimuli in the brain. AdoMetDC activity has been reported to decrease after traumatic injury (Henley et al. 1997) and cerebral ischemia (Rohn et al. 1992), increase in epilepsy (Rohn et al. 1992, Morrison et al. 1994) and in Alzheimer’s disease (Morrison et al. 1993b), and remain unchanged after restrained stress (Gilad & Gilad 1996). The induction of ODC activity is most pronounced following severe metabolic stress as produced by cerebral ischemia (Paschen et al. 1993). Induction of ODC and concomitant decrease in AdoMetDC activity brings about a high increase in the putrescine level. There is a single report on elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury (Rao et al. 2000), which suggests that SSAT activity is increased as well and may contribute to the putrescine levels. The levels of spermidine and spermine do not change significantly or they can decrease temporarily (Paschen et al. 1993). The role of increased putrescine concentration is still unclear. In some studies, excessive polyamines have been implicated in neuronal degeneration after CNS injury (Paschen et al. 1993, Schmitz et al. 1993, Kindy et al. 1994, Baskaya et al. 1997, Dogan et al. 1999a, Dogan et al. 1999b). On the other hand, it has been suggested that the postischemic activation of polyamine metabolism is necessary for the recovery of neurons from metabolic stress because the pretreatment of animals with polyamines reduces the postischemic development of neuronal necrosis (Gilad & Gilad 1991). This was supported by observation that after cerebral ischemia ODC is up-regulated also in regions where no cellular damage usually occurs (Keinänen et al. 1997) and studies on transgenic rats overproducing ODC have demostrated that induction of ODC has neuroprotective role in this model of transient focal cerebral ischemia (Lukkarinen et al. 1997, Lukkarinen et al. 1998).
A few histochemical studies on the expression patterns of ODC and antizyme in CNS have been published. Studies on ODC have focused mainly on the developmental pattern of expression or on the effect of pathological stimuli on expression. Polyamines themselves have also been localized employing immunocytochemical methods. Detectable ODC expression appears to be restricted to neurons (Müller et al. 1991, Müller et al. 1993, Bernstein & Müller 1995, Ichikawa et al. 1997, Ichikawa et al. 1998, Gritli-Linde et al. 2001), glial expression of ODC has been reported only in few cases (Bernstein & Müller 1999). These reports have clearly stated that ODC is a cytoplasmic protein. Antizyme expression is similarly confined to neurons as demostrated both by immunocytochemistry and in situ hybridization (Junttila et al. 1995, Gritli-Linde et al. 2001). Antizyme is mainly localized to the cytoplasm, but as shown in this thesis (III) and later by others (Gritli-Linde et al. 2001) it can be found in certain cell types or developmental stages predominantly or even virtually exclusively in nuclei. Although glial cells do not contain detectable amount of ODC, immunocytochemical studies with antibodies against spermidine and spermine have shown that polyamine levels in these cells are comparable to neurons and even higher in some cases (Laube & Veh 1997, Biedermann et al. 1998, Skatchkov et al. 2000). It has been suggested that the glial cells could be a polyamine storage (Laube & Veh 1997), but it is not known how their high polyamine concentration is formed and maintained.
2.1.5. Polyamines and the regulation of inward rectifying potassium channels
Inwardly rectifying potassium (Kir) channels have two main physiological roles: they stabilise the resting membrane potential near the K+ equilibrium potential and they mediate K+ transport across membranes (for reviews, see Williams 1997, Reimann & Ashcroft 1999, Oliver et al. 2000). Kir channels conduct more current when the membrane potential is hyperpolarized than when it is depolarized from the K+ equilibrium potential by an equivalent amount, i.e. Kir channels favour the inward flow of potassium ions. This property results principally from a voltage-dependent block of outward currents by cytoplasmic Mg2+ and polyamines that enter the pore under the influence of the membrane voltage field and impede K+ efflux.
There are seven subclasses of Kir channels of which Kir2 channels (Kir2.1 – Kir2.4) represent typical “strong” rectifiers. The term strong refers to the high voltagedependence of rectification. Polyamines, mainly spermine, are responsible for the intrinsic gating and rectification of Kir2 channels (Fakler et al. 1994, Ficker et al. 1994, Lopatin et al. 1994, Fakler et al. 1995) and Kir3 channels (Yamada & Kurachi 1995). The latter belong to a subfamily of G-protein-activated intermediate rectifiers. Polyamines have been demonstrated to rectify also ATP-dependent Kir4.1 channel (Fakler et al. 1994, Fakler et al. 1996) and weak Kir6.2 channels in alkaline pH (Baukrowitz et al. 1999). Weak rectifyers of Kir1 subfamily are relatively insensitive to polyamines (Fakler et al. 1994, Yamada & Kurachi 1995, Riochet et al. 2001).
Members of Kir2 subfamily are typically expressed in heart, skeletal muscle and nervous system. Another subfamily expressed widely in CNS is Kir3, in addition to central neurons they are found in a variety of tissues and cell types, among them cardiac myocytes (Oliver et al. 2000). Kir4.1 is expressed in glial cells and may be their predominant Kir channel (Takumi et al. 1995, Hibino et al. 1999, Kofuji et al. 2000). Rather ubiquitous Kir6 channels are expressed also in neurons of the CNS (Liss & Roeper 2001). The significance of Kir channels for the normal function of cells is confirmed by observations that their genes are mutated in three human and one murine hereditary diseases. Mutation in Kir1.1 leads to Bartter’s syndrome type III (Simon et al. 1996). The Kir2.1 channel is mutated in Andersen’s syndrome (Plaster et al. 2001) and Kir6.2 in familial persistent hyperinsulinaemic hypoglycaemia of infancy (Thomas et al. 1996) whereas knock-out mice lacking Kir6.2 channel are susceptible to generalized seizures after brief hypoxia (Yamada et al. 2001). Mutation in the pore region of Kir3.2 results in weaver phenotype of mice (Patil et al. 1995). Bartter’s syndrome is a renal tubular disorder characterised by salt-wasting, hypokalaemia and metabolic acidosis. Andersen"s syndrome is a rare disorder characterized by periodic paralysis, cardiac arrhythmias, and relatively severe dysmorphic features. Weaver mice have selectively lost brain neurones and they suffer from ataxic gait.
Sequence comparison between the prototypes of strong and weak rectifiers, Kir2.1 and Kir1.1 has led to identification of three residues defining the voltage-dependence of polyamine block: negatively charged glutamate or aspartate residues in the second transmembrane segment (TM2 site) (Fakler et al. 1994, Lu & MacKinnon 1994, Stanfield et al. 1994, Wible et al. 1994) and in two sites of the cytoplasmic C-terminus (Taglialatela et al. 1995, Yang et al. 1995, Kubo & Murata 2001). Exchange of the neutral TM2 site in the weakly rectifying Kir1.1 to a negatively charged residue converted this channel into a strong inward-rectifier (Wible et al. 1994), while neutralizing TM2 site and any of C-terminal sites in Kir2.1 greatly reduced spermine-mediated rectification (Taglialatela et al. 1995, Kubo & Murata 2001). The requirement of negatively charged amino acid at the TM2 and C-terminal sites for strong rectification suggests electrostatic interactions between these sites and the positively charged blocker molecules (Oliver et al. 2000). This is further supported by findings that a reporter cysteine at the TM2 site of the transmembrane segment can be readily modified with cysteine-reactive reagents indicating exposure of TM2 site to the lumen of pore (Lu et al. 1999). Kir6.2 displays polyamine-responsive rectification only in alkaline pH. This pH-dependency has been shown to be confered by C-terminal histidine. Around neutral pH, the histidine is protonated and Kir6.2 channels present weak inward-rectification, while alkalization that deprotonates the residue results in strongly rectifying channels (Baukrowitz et al. 1999). This pH-dependent rectification might in metabolic stress inhibit electrical activity and help protect the cell from energy depletion.
It has been shown using inhibitors of polyamine biosynthesis and ODC-deficient CHO cell line that changes in cellular polyamine contents can indeed alter the rectification of Kir channels and the excitability of cells (Bianchi et al. 1996, Shyng et al. 1996). The effect of elevated polyamine levels on Kir channels has been studied using transgenic mice that overexpress ODC in the heart under control of the cardiac α-myosin heavy chain promotor (Lopatin et al. 2000). In transgenic hearts putrescine levels were elevated 35-fold, the spermidine level was increased 3.6-fold, but spermine was essentially unchanged. Despite these changes, alterations in Kir currents of trangenic cardiomyocytes were relatively small. Inward rectification as well as voltage dependence of rectification were essentially unchanged although density of current was reduced. In cardiomyocytes isolated from Gyro (Gy) mice that are deficient in spermine synthase (Lorenz et al. 1998, Meyer et al. 1998) and totally lacking spermine but displaying increased (5.4-fold in the heart) spermidine levels, inward rectification of Kir channels was slighly reduced (Lopatin et al. 2000), but the density of Kir currents was unchanged. Direct manipulation of polyamine levels in myocytes by pipette dialysis indicated that spermidine and especially spermine are major controllers of rectification in intact cells. Putrescine plays relatively little role in controlling, but may cause significant weakly voltage-dependent block. The ODC transgenic and Gy mice are apparently normal, and only relatively minor effects on action potential characteristics and heart rates can be expected. Both cardiac hypertrophy (Calderera et al. 1974, Bartolome et al. 1980) and epilepsy (Laschet et al. 1992, Hayashi et al. 1993, Mialon et al. 1993) are each associated with increased polyamine levels and with enhanced excitability, but it is not known whether these two things are related to each others.
2.1.6. Polyamines and cancer
In cancer cells and tissues polyamine levels and polyamine biosynthesis are highly elevated (Scalabrino & Feriolo 1982, Pegg 1988, Marton & Pegg 1995). ODC has been the main focus of interest, but many human cancers show also increased AdoMetDC activity, although to a lesser extent than ODC activity. ODC becomes activated after treatment with chemical carcinogens and tumor promotors, as well as in cells transformed by various oncogens, such as v-src, neu and ras (Hölttä et al. 1988, Pegg 1988, Sistonen et al. 1989a, Sistonen et al. 1989b, Auvinen et al. 1992). Furthermore, the overexpression of normal human ODC in NIH3T3 or RAT-1 fibroblasts induces malignant transformation including the ability to grow as colonies in semi-solid medium and to form rapidly progressing tumors in nude mice (Auvinen et al. 1992, Moshier et al. 1993, Auvinen et al. 1997). In another study overexpression of ODC was not able to transform NIH3T3 cells without co-expression of c-Ha-ras oncogene (Hibshoosh et al. 1991). Cells overexpressing ODC were more readily transformed by c-Ha-ras than wild-type NIH3T3 cells. Also overexpression of AdoMetDC can lead to malignant transformation of NIH3T3 cells (Paasinen-Sohns et al. 2000). Interestingly, AdoMetDC appeared to be an even more potent inducer of transformation than ODC.
A possible role for ODC in carcinogenesis was suggested for the first time based on a series of studies carried out using a skin-tumor model initiated by a carcinogen (initiator) and promoted by phorbol ester (O"Brien et al. 1975a, O"Brien et al. 1975b). These treatments resulted in high induction of ODC activity. Although cells tranformed by overexpression of ODC (Auvinen et al. 1992) or AdoMetDC (Paasinen-Sohns et al. 2000) were able to form tumors in nude mice, the first transgenic mice overexpressing ODC did not show increased levels of spontaneous tumorigenesis during their entire lifetime (Alhonen et al. 1995) and were actually able to maintain normal levels of polyamines in their tissues (Halmekytö et al. 1991a, Halmekytö et al. 1993). However, they displayed enhanced papilloma formation in response to chemical skin tumor promotion (Halmekytö et al. 1992). Similar observations were made with K6/ODC transgenic mice that overexpressed ODC in hair follicle keratinocytes (O"Brien et al. 1997) and these mice display a slightly increased sensitivity even to spontaneous tumors (Megosh et al. 1995). A single low dose of carcinogen was generally enough to initiate tumorigenesis in the K6/ODC mice. They often harboured mutations to c-Ha-ras gene after administration of initiating doses of carcinogens (Megosh et al. 1998). When the K6/ODC transgenic mice were bred with TG.AC v-Ha-ras transgenic mice, double transgenic mice produced spontaneously skin carcinomas (Smith et al. 1998). Their spontaneous tumors were reversed by ODC inhibitor DFMO (Lan et al. 2000). Similar kinds of results were obtained when cells infected with ODC or ras construct or with both were transferred to nude mice (Clifford et al. 1995). Only cells infected to overproduce both ODC and c-Ha-ras oncoprotein were able to form tumors. The K6/ODC mice – now called K5/ODC mice – have recently been shown to be very sensitive to UVB radiation (Ahmad et al. 2001). After 30 weeks of repeated exposure to UVB, 40 % of the K5/ODC mice were found to develop epidermal tumors, whereas SKH-1 hairless mice, the most common and highly sensitive model for photocarcinogenesis (Bickers & Athar 2001), did not develop tumors for up to 50 weeks. The central role of ODC in epidermal carcinogenesis has been even further emphasized by the observation that the targeted expression of ODC inhibitor antizyme in the keratinocytes of transgenic mice decreased sensitivity to chemical carcinogenesis (Feith et al. 2001).
Tumor cells may also have a specific ODC form or specific post-translational modification or regulation of ODC may take place, because ODC activity in some tumors is activated by GTP (O"Brien et al. 1986, O"Brien et al. 1987, Hietala et al. 1988), normally a property only of some bacterial ODCs. Tumor specific GTP-activatable ODC is discussed in more detail later in the chapter 2.2.4.
It is not clearly understood how forced overexpression of ODC or AdoMetDC leads to malignant transformation. The involvement of some components of common signal transduction pathways has been shown. Transformation by ODC overexpression resulted in phosphorylation of Ras nucleotide exchange factor Sos-1, Raf-1 kinase and c-Jun that forms a transcription factor AP-1 as a dimer with c-Fos and regulates transcription of target genes (Paasinen-Sohns & Hölttä 1997). Interestingly, Erk1 and Erk2 kinases of the MAP family were not needed to mediate signal. Similarly, c-Jun appeared to be the integral mediator of signaling in transformation induced by AdoMetDC overexpression (Paasinen-Sohns et al. 2000). Transformant displayed constitutive activation of the c-Jun NHacetylspermidine-terminal kinase (JNK) pathway. The expression of dominant-negative mutants of JNK1 and SEK1, both kinases upstream from c-Jun, reverted the phenotype of the AdoMetDC transformants. At the level of cell cycle regulation, the largest common effect in the ODC and AdoMetDC transformants was the constitutive down-regulation of cyclin-dependent kinase inhibitor p27Kip1 and its loss from the cyclinE/cyclin-dependent kinase 2 complexes (Ravanko et al. 2000). In addition, the level of cyclin D1 and cyclin D1-dependent kinase as well as total cyclin dependent kinase 4 activities were elevated in both transformants suggesting that the expression of ODC or AdoMetDC may effect on cell cycle regulation in many ways.
With high levels of polyamines being so strongly associated with rapid proliferation and growth of tumors, it is understandable that great effort has been placed on designing inhibitors of polyamine synthesis and polyamine analogs and testing their ability to restrict growth of tumor and cancer cells. One of the first inhibitors, and maybe the most studied, is α-difluoromethylornithine, an irreversible inhibitor of ODC (Bey et al. 1978, reviewed in Meyskens & Gerner 1999). Treatment of mammalian cell cultures, rodents, or humans with DFMO generally causes a suppression of putrescine and spermidine contents in cells and tissues in which intracellular polyamine pools depend on ODC activity, without affecting spermine levels (Gerner & Mamont 1986, Pegg 1988, Meyskens et al. 1998). Inhibition of ODC has been found to suppress tumor formation in experimental models of bladder (Nowels et al. 1986), breast (Thompson et al. 1986), intestinal (Nigro et al. 1986) and skin carcinogenesis (Peralta Soler et al. 1998, Arbeit et al. 1999).
However, early clinical cancer therapeutic trials with DFMO were disappointing, and at high doses several side effects have occurred including diarrhea, abdominal pain, moderate anemia and temporal loss of hearing (Abeloff et al. 1984, Abeloff et al. 1986, Talpaz et al. 1986, Harari et al. 1990). DFMO has been later observed to have a considerable effect on recurrent gliomas (Levin et al. 1992). On the other hand, in phase III trial, no benefit of DFMO was seen in the treatment of glioblastoma multiforme when tested together with accelerated hyperfractionation or standard fractionated radiotherapy (Prados et al. 2001). Currently DFMO is mainly studied and tested as a chemoprevention agent (see e.g. Love et al. 1993, Meyskens et al. 1994, Mitchell et al. 1998b, Carbone et al. 2001, Simoneau et al. 2001). At low doses proposed for long-term chemoprevention trials, no systematic side effects have been seen. Trials are at the stage were suitable doses are determined and effect of DFMO on tissue polyamine levels tested.
Other polyamine synthesis inhibitors tested in clinical trials are AdoMetDC inhibitors methylglyoxal bis(guanylhydrazone) (MGBG) (Warrell & Burchenal 1983, Herr et al. 1986) and SAM486A (CGP 48664) (Paridaens et al. 2000). MGBG is toxic if doses are high, in combination with DFMO it has shown some effect against gliomas (Levin et al. 1992). SAM486A has displayed promising antiproliferative activity in cell cultures (Regenass et al. 1994, Manni et al. 1995, Mi et al. 1998) and preclinical animal studies (Paridaens et al. 2000).
Various investigators have synthesized structural analogues that can be taken into cells by the polyamine transport system and that can mimic the natural polyamines by downregulating synthetic or inducing catabolic pathways, but that are unable to substitute for polyamines in terms of supporting cell growth and differentiation (reviewed in Casero & Woster 2001). Most analogues are symmetrically or unsymmetrically alkylated derivatives of polyamines. Alkyl groups are generally added to terminal primary amino groups. Many of analogues have displayed some antitumor activity in cancer cell lines (Porter et al. 1987, Porter et al. 1991, Chang et al. 1992, Davidson et al. 1993) and in xenografts of human tumors (Bernacki et al. 1995, Sharma et al. 1997). Perhaps the most successful alkylpolyamine to date is N1-N11-diethylnorspermine (DENSPM). It has been tested in phase 1 clinical trial for doses (Creaven et al. 1997, Streiff & Bender 2001) and is currently undergoing phase II trial (Casero & Woster 2001). It induces highly SSAT activity that leads to decrease in cellular polyamine levels (Gabrielson et al. 1999).