Polyamines: from molecular biology to clinical applications.

Trends in Molecular Medicine

Polyamines: From Molecular Biology to Clinical Applications

Ann Med Downloaded from informahealthcare.com by York University Libraries on 03/04/15 For personal use only.

Juhani Janne', Leena Alhonen' and Pekka Leinonen2

The polyamines putrescine, spermidine and spermine represent a group of naturally occurring compounds exerting a bewildering number of biological effects, yet despite several decades of intensive research work, their exact physiological function remains obscure. Chemically thesecompoundsareorganicaliphaticcations with two(putrescine), three (spermidine) or four (spermine) amino or imino groups that are fully protonated at physiological pH values. Early studies showed that the polyamines are closely connected to the proliferation of animal cells. Their biosynthesis is accomplished by a concerted action of four different enzymes: ornithine decarboxylase, adenosylmethionine decarboxylase, spermidine synthase and spermine synthase. Out of these four enzymes, the two decarboxylases represent unique mammalian enzymes with an extremely short half life and dramatic inducibility in response to growth promoting stimuli. The regulation of ornithine decarboxylase, and to some extent also that of adenosylmethionine decarboxylase, is complex, showing features that do not always fit into the generally accepted rules of molecular biology. The development and introduction of specific inhibitors to the biosynthetic enzymes of the polyamines have revealed that an undisturbed synthesis of the polyamines is a prerequisite for animal cell proliferation to occur. The biosynthesis of the polyamines thus offers a meaningfultarget for the treatment of certain hyperproliferative diseases, most notably cancer. Although most experimental cancer models responds strikingly to treatment with polyamine antimetabolites - namely, inhibitors of various polyamine synthesizing enzymes - a real breakthrough in the treatment of human cancer has not yet occurred. It is, however, highly likely that the concept is viable. An especially interesting approach is the chemoprevention of cancer with polyamine antimetabolites, a process that appears to work in many experimental animal models. Meanwhile, the inhibition of polyamine accumulation has shown great promise in the treatment of human parasitic diseases, such as African trypanosomiasis. Key words: putrescine; spermidine; spermine, o r n i t h i n e decarboxylase; adenosylmethionine decarboxylase; difluoromethylornithine; mitoguazone. (Annals of Medicine 23: 241-259,1991) The history of the polyamines is long, the first written document about the existence of spermine crystals in human semen dating back to 1678 (1). Paradoxically, although more than 300 years have elapsed since that observation no one can definitively describe the function of seminal spermine. The natural polyamines include, in addition to the tetraamine spermine, a diamine putrescine and a triamine spermidine. As Figure 1 shows the structures of the polyamines are relatively simple, representing organic amines

with aliphatic carbon chain. The fact that these compounds are positively charged at physiological pH values and hence exert high affinity to any negatively charged cellular components makes it hard to elucidate their possible cellular functions besides a straightforward cation effect. As will be shown later, however, these compounds are indispensable for cell growth irrespective of their exact modes of action.

Biosynthesis of the Polyamines From the Department of Biochemistry & Biotechnology, University of Kuopio and 'Departments of Gynecology and Obstetrics, Helsinki University Central Hospital. The experimental work in the authors' laboratory has received financial support from the Academy of Finland and from the Finnish Foundation for Cancer Research. Address and reprint requests: Juhani Janne, M.D., Department of Biochemistry & Biotechnology, University of Kuopio, P.O. Box 1627, SF-70211 Kuopio, Finland.

As shown in Figure 1, the primary carbon and nitrogen sources for putrescine, spermidine and spermine are the amino acids L-methionine and L-ornithine. In animal cells the latter compound is formed from L-arginine in a reaction catalysed by arginase. Even though arginase is not usually included as a real polyamine biosynthetic enzyme, recent experimental evidence suggests that some forms of arginase exclusively function to generate ornithine for the

Janne Alhonen Leinonen

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NHzCH#H&H$HCH&HzCH&H,NHCH&H2CH~~ SPERMINE Figure 1. Biosynthesisof putrescine, sperrnidine and sperrnine from anginine and rnethionine. The reactions are catalysed by the following enzymes: 1, arginase; 2, ornithine decarboxylase; 3, S-adenosylrnethioninesynthetase; 4, S-adenosylmethioninedecarboxylase; 5, spermidine synthase; 6, sperrnine synthase.

biosynthesis of the polyamines. Ornithine is converted to putrescine in a seemingly simple decarboxylation reaction catalysed by ornithine decarboxylase. The regulation of the release of carbon dioxide from ornithine is, however, far from simple. Putrescine serves as a precursor for spermidine synthesis, being coupled to a propylamine moiety derived from decarboxylated adenosylmethionine. The latter compound is generated by the action of adenosylmethionine decarboxylase on adenosylmethionine, a compound which is better known as a methyl donor in biological methylations. Adenosylmethioninedecarboxylase shares common properties with ornithine decarboxylase. The coupling of putrescine to the propylamine group to yield spermidine is catalysed by spermidine synthase. Similar coupling of spermidine to the same propylamine moiety to yield spermine is catalysed by spermine synthase, which resembles spermidine synthase but is an entirely different enzyme. All the reactions described above are irreversible in practice. Thus to convert spermine back to putrescine, which is what occurs in a living mammalian cell, a completely different set of enzymes is required. In brief, spermine or spermidine is first acetylated by a polyamine acetylase and oxidised by a polyamine oxidase to yield spermidine from spermine and putrescine from spermidine. Although in many instances these enzymes required for the backconversion may be critical, we will be concentrating on the biosynthetic enzymes. So let us look more closely at the individual enzymes engaged to the biosynthesis of the polyamines.

Ornithine Decarboxylase This unique mammalian enzyme was discovered in 1968 simultaneously and independently in three laboratories,

two in the United States (2, 3) and one in Finland (4). Associated with the discovery of this enzyme was a finding that suggested that ornithine decarboxylase may be one of the most inducible mammalian enzymes (2, 4). After its discovery ornithine decarboxylase soon became one of the most studied of mammalian enzymes. At present more than 1000 research reports have been published describing various features of this enzyme. Ornithine decarboxylase is a unique mammalian enzyme in many respects. The first puzzling observation was the fact that ornithine decarboxylase turns over very rapidly, its half life being less than 0.5 h (5). This may be a meaningless figure for many readers, but placing it in perspective, the average half life of a mammalian enzyme is counted in days rather than in minutes. After many years of pure phenomenology, mammalian ornithine decarboxylase was subsequently purified from a variety of sources (6). It turned out that by any definition this enzyme is a low abundance protein; representing only about 3 ppm of the soluble proteins of a mammalian cell. This explains why ornithine decarboxylase was not cloned and its amino acid sequence determined from cDNA until 1984. Once again three laboraties simultaneously accomplished the cloning of mouse ornithine decarboxylase (7-9). At first sight, the amino acid sequence of ornithine decarboxylase did not reveal any "unusual" features in its primary structure. The enzyme contains a high number of cysteine residues that may account for the fact that thiol reducing agents are needed to maintain enzymatic activity (10, 11). As to the rapid intracellular degradation of the enzyme, a unique feature of the amino acid sequence of ornithine decarboxylase may offer an explanation. Mammalian ornithine decarboxylase, together with a family of short half-life proteins, contains regions rich in proline (P), glutamic acid (E), serine (S) and threonine


Polyamines: From Molecular Biology to Clinical Applications (T), called PEST sequences (12). These PEST regions have been found in almost all proteins with a half life of less than 0.5 h (12). Interestingly, in Trypanosoma brucei, a parasite causing African sleeping disease, ornithine decarboxylase is deprived of one of the PEST regions and indeed it turns over much more slowly than in its mammalian counterpart. More convincingly, a partial truncation (including a PEST sequence) of the mouse ornithine decarboxylase at its carboxy terminus results in a striking stabilisation of the enzyme (13). The isolation of human ornithine decarboxylase cDNA from a liver cDNA library and the subsequent sequencing of it showed close identity between murine and human enzyme, both at the nucleotide and at the amino acid levels (1 4). The similarities between mammalian ornithine decarboxylases do not involve only the structure of the protein (or the mRNA), but the structure and general organization of the genes in mouse (1 5 , 16), rat (17, 18) and man (19-22) likewise are remarkably similar. The active gene of human ornithine decarboxylase resides at the short arm of chromosome 2 (23). Interestingly, the corresponding mouse gene resides at chromosome 12, which is the murine equivalent to the short arm of human chromosome 2 (24). In addition to the active sequences at chromosome 2, human genome contains a processed pseudogene at chromosome 7 (22, 23). As shown in Figure 2, two other interesting mammalian genes are located in the close proximity of human active ornithine decarboxylase gene at chromosome 2; the gene for the catalytic subunit (M2) of ribnucleotide reductase, a key enzyme in the synthesis of DNA, and the N-myc oncogene. The gene for ornithine decarboxylase is coamplified with the M2 gene (in hydroxyurea resistant tumour cells) (25) and also occasionally in human tumours with amplified Nmyc sequences (26).

Adenosylmethionine Decarboxylase Many similarities occur between ornithine and adenosylmethionine decarboxylases. With enzymes turning over unusually rapidly in the living cell, the half life of adenosylmethionine decarboxylase is something between 30 and 60 min (27). Much of the molecular genetics of this enzyme is likewise elucidated. Its amino acid sequence has been deduced for both mouse and human (28) as is its conversion from a proenzyme to an active enzyme (29). Unlike most mammalian decarboxylases, adenosylmethionine decarboxylase does not utilise pyridoxal phosphate as the coenzyme but instead a covalently bound pyruvate undertakes the coenzyme function (30, 31 ) . Athough adenosylmethionine decarboxylase can also be classified as an inducible enzyme, its inducibility is not nearly as dramatic as that of ornithine decarboxylase.

Spermidine and Spermine Synthases In contrast to the two decarboxylases described the enzymes spermidine and spermine synthases are stable proteins that occur in much larger amounts than decarboxylases (27). Their regulation appears to be determined mainly by the availability of the substrate decarboxylated adenosylmethionine provided by adenosylmethionine decarboxylase. Spermidine synthase activity is found both in prokaryotes and eukaryotes, yet spermine synthase appears to be the enzyme of the eukaryotes (32). Although

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HUHAN CHROllOSOtlE2 Figure 2. Human chromosome 2 and the localisation of some genes at it. ODC, ornithine decarboxylase; FIR, ribonucleotide reductase; TGF-a, transforming growth factor a ; IGK, mrnunoglobuiin kappa.

catalysing almost identical reactions, these two synthases have structurally very little in common (33). Human spermidine synthase has recently been cloned and its amino acid sequence has been determined from the cDNA (34). The active gene appears to be located at human chromosome 1 (unpublished observations).

What Do the Polyamines Do? Polyamines are organic cations that are positively charged at physiological pH values. Thus they have the inherent property to react strongly with negatively charged components of a living cell. Hence the biological effects obtained by adding polyamines in vitro may have nothing to do with their physiological function in a living cell. In fact, nobody has ever measured the concentrations of free polyamines in the cellular compartments indeed, it is probably impossible to do that. About 800 research reports have been published describing various effects exerted by polyamines in various experimental systems. Keeping in mind the critical limitations mentioned above, the value of those reports is limited. As expected, the polyamines interact with whole cells, cell organelles, nucleic acids, finally influencing the rate of innumerable metabolic reactions. Pharmacologically the polyamines, especially spermidine and spermine, are toxic substances. The toxicity is manifested as nephrotoxicity, hypothermia and sedation (27). One of the most exotic actions ascribed to the polyamines is the initiation of the burial of dead conspecifics in rats (35). This phenomenon suggest that the polyamines may act as pheromone like substances in rodents. To draw any conclusions about the physiological function of the polyamines based on the biological effects exerted by them is an impossible task because there are too many reported effects and they are hopelessly diffuse. Two approaches are still available: (i) the use of polyamine auxotrophic animal cells, and (ii) a production of polyamine depletion by means of specific chemical intervention of their biosynthesis. Even these approaches however, have

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inherent limitations as it is extremely difficult to judge what the primary effect is in response to polyamine deprivation. Based on the use of these two approaches, the fact has been established that undisturbed formation of the polyamines is essential for animal cell proliferation to occur. The consequences of the inhibition of polyamine biosynthesis in cultured animal cells as well as in several tumour bearing animal models are described in detail on page 245.

obtaining comparable tissue samples. In any event, some indirect experimental evidence suggests that an enhanced ornithine decarboxylase activity, such as might result from gene amplification, may give distinct growth advantage to tumour cells as manifested by an enhanced aggressiveness (47) or as an ability to form colonies in semisolid media (48). Recently, we have extended this approach by generating transgenic mice carrying human ornithine decarboxylase gene and expressing it in a highly aberrant way in almost all tissues studied (49).

Polyamines and the Growth of Animal Cells

Expression of Human Ornithine Decarboxylase is Influenced by Gene Methylation

Following the discovery of mammalian ornithine decarboxylase in regenerating rat liver (2, 4) it became evident that an early accumulation of putrescine and spermidine was a sign of accelerated growth (27). It now seems that the initiation of the growth process in animal cells almost always involves a striking stimulation of ornithine decarboxylase.

Ornithine Decarboxylase Is a Growth-Related Enzyme Some ornithine decarboxylase activity is probably required for any living cell to maintain its metabolic activity. During cell proliferation, however, this enzyme apparently confers a distinct growth advantage on the cell. Ornithine decarboxylase responds rapidly to a wide variety of both anabolic and catabolic stimuli by increasing or decreasing its activity. The mechanisms involved in regulating the enzyme are complex. A straightforward transcriptional activation - the most economical way to regulate the steady state levels of a given protein -although operating (6),is apparently not the major regulatory mode for this enzyme. In most instances the expression of the enzyme is regulated posttranscriptionally, possibly at the translational level (3641) or even at some post-translational level (42). Transfection experiments have shown that the short half life of ornithine decarboxylase has a major impact on its expression. Transfection of the promoter region of mouse ornithine decarboxylase gene fused to various reporter genes and the subsequent stimulation of the transfectant cells has shown that the promoter region may not primarily govern the expression of the enzyme (42). We have obtained similar results using the human promoter region fused with the firefly luciferase gene (unpublished results). Thus it appears that the short half life is critical for rapid stimulation of the enzyme. To support this view, Coffin0 and his coworkers (13) showed that stabilisation of mouse ornithine decarboxylase by cleaving off part of the carboxy terminus greatly reduced the inducibility of the enzyme (13). Also, mathematical models, extending back to late seventies (43) and specifically applied to ornithine decarboxylase (44), show that a rapid rate of degradation is directly related to the inducibility of a given enzyme whenever the protein synthesising machinery is generally activated. Although numerous reports (27, 45, 46) suggest that ornithine decarboxylase activity is higher in malignant and transformed cells than in normal cells, any systematic analyses have been greatly hampered by the rapid fluctuations in enzyme activity as well as by the difficulties in Ann Merl7.q

In the mammalian genome it is generally accepted that the methylation of a given gene, most notably at CpG sites (cytosine methylation), influences its expression rate (5053). In most cases the methylation (most often in the promoter region) of a gene inhibits or even abolishes its expression (50). The molecular mechanisms responsible for the methylation induced inhibition of gene expression are still poorly known. Many possibilities exist. Gene methylation probably affects the local conformation of DNA (54), cytosine methylation may render parts of the gene inaccessible to various transcription factors required for the proper expression of the gene (51, 55), or more likely there are specific proteins bound to the methylated parts of the gene thus preventing the binding of positive transcription factors at responsive gene elements (55, 56). Finally, DNA methylation appears to be involved in parental imprinting of a given gene (57-59). In our own work on cultured human myeloma cells we found that these cells were sensitive to glucocorticoids which exerted antiproliferative effects associated with an inhibition of ornithine decarboxylase. A gradual adaptation of the myeloma cells to increasing concentrations of glucocorticoids led to the development of resistance to the corticoids. Interestingly, this resistance was associated with an enhanced accumulation of ornithine decarboxylase mRNA and disappearance of the inhibition of the enzyme activity. The most likely explanation for this phenomenon was a distinct hypomethylation of genomic sequences for ornithine decarboxylase (60). We found subsequently that different human cell lines showed great variations in the methylation status of ornithine decarboxylase gene as the human myeloma cells seemed to be heavily methylated in comparison with certain leukaemia cell lines (61). These studies were subsequently extended to a few patients with blood malignancies. The results showed that ornithine decarboxylase gene hypomethylation was confined to chronic lymphatic leukaemia and, interestingly, among several oncogenes studied similar hypomethylation was also found in genomic sequences of the thyroid hormone receptor like protooncogene erb-A1 (62). Re-examination of the dexamethasone resistant human myeloma cells with ornithine decarboxylase gene hypomethylation showed that similar hypomethylation had occurred likewise in erb-A1 proto-oncogene (unpublished results). We also used isolated and cloned human ornithine decarboxylase gene (derived from human myeloma cells with gene amplification) and showed that a methylation in vitro of the gene abolished any transient expression of ornithine decarboxylase after transfection (63). A hypomethylation of ornithine decarboxylase gene may thus transcriptionally activate the

Polyamines: From Molecular Biology to Clinical Applications expression of the enzyme under various pathophysiological circumstances.


Inhibition of Ornithine Decarboxylase Leads fo Growth Arrest of Animal Cells The precise cellular function of the polyamines remains obscure despite intensive research efforts over many decades. It is, however, beyond doubt that the polyamines somehow participate in the process of cellular proliferation. This conclusion may be reached from many experimental studies in which highly specific inhibitors of the biosynthetic enzymes of the polyamines were used. Ornithine decarboxylase obviously served as a logical target for chemical intervention as the enzyme appeared to be closely connected with cell proliferation. The selection of ornithine decarboxylase as the major target for chemical intervention was based on several experimentally verified facts. (i) The activity of ornithine decarboxylase is under most circumstances, though admittedly not always, the lowest among the enzymes engaged with the synthesis of the polyamines. (ii) Putrescine, the product of the reaction, is required for the activation of adenosylmethionine decarboxylase (64, 65). (iii) Finally, putrescine is needed for the processing of adenosylmethionine decarboxylase peptide to the active enzyme (29, 66). Design, search and synthesis of inhibitors for ornithine decarboxylase began soon after the discovery of the enzyme activity in mammalian tissues. Although in the early nineteen seventies, many promising inhibitors were found such as competitive inhibitors, compounds acting through the coenzyme pyridoxal phosphate or indirectly acting diamine inhibitors (for references see 27), a real breakthrough in this area occurred in 1978 when Metcalf et al. (67) succeeded in synthesising a-difluoromethylornithine, a derivative of the amino acid ornithine. This remarkable compound belongs to the so called mechanism based inhibitors or suicide inhibitors. As implied by the mechanism of action, difluoromethylornithine (DFMO or eflornithine) serves as a real substrate for ornithine decarboxylase and is, in fact, decarboxylated during the reaction. As a result of the decarboxylation, however, a series of electron rearrangements occurs leading to an irreversible inactivation of ornithine decarboxylase owing to an alkylation of its active centre. Based on the mechanism of action, DFMO is entirely specific to ornithine decarboxylade and does not interfere with any other enzymatic reactions using ornithine as the substrate. Since the introduction of DFMO, nearly 700 papers have been published mainly describing its use as an antiproliferative agent. Exposure of rapidly growing animal cells to DFMO results in a fast and virtually complete depletion of putrescine and spermidine while the concentration of spermine usually rises a little (10, 68). The absence of putrescine and sperrnidine - spermine remains at near normal concentration - seems to be sufficient to result in a reversible cytostatic effect in most studies of animal cell lines so far. A few human-derived cell lines, such as small cell lung carcinoma and HeLa cells, however, respond to DFMO in a cytocidal fashion (69, 70). The cytostatic, rather than cytocidal, nature of the antiproliferative action exerted by DFMO has often been attributed to the fact that the drug does not affect the pool of spermine (71). Even though some experimental evidence is available suggesting that transformed cells behave differently as re-

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gards their cell cycle traverse in response to inhibitors of ornithine decarboxylase (72), it has become increasingly evident that this may not be a general rule (73). Putrescine and spermidine depletion as achieved with inhibitors of ornithine decarboxylase has quite opposite effects on differentiation, depending on the cell line used. Thus murine embryonal carcinoma cells (74-76), mouse melanoma cells (77-79), hematopoeitic precursor cells (80) and mouse neuroblastoma cells (81) start to differentiate, as judged by the expression differentiated phenotypic markers, in response to the inhibition of ornithine decarboxylase. On the contrary, in some cell types polyamine limitation has been shown to inhibit the expression of differentiated phenotype induced by various chemicals. These cells include Friend erythroleukaemia cells (82), human promyelocytic leukaemia cells (83), myoblasts (84) and 3T3-L cells (85, 86). It is hard to explain logically this contrasting behaviour of these cells in response to the inhibition of ornithine decarboxylase. The only imaginable difference between these experimental models was the fact that polyamine limitation appears to inhibit chemically or hormonally induced differentiation, whereas the stimulation of differentiation occurred solely in response to the inhibition of ornithine decarboxylase. In addition to DFMO, many other mechanism based inhibitors, either derivatives of ornithine or putrescine, have been subsequently synthesised and tested in various experimental systems (87). Although in many instances more potent on a weight basis or because they enter more easily into the cells, these new inhibitors have added little to the experience obtained with DFMO which still remains as the inhibitor of choice. The old “indirect diamine inhibitor” approach has been subsequently followed by the introduction of certain alkylated derivatives of spermidine and spermine, such as bis(ethy1)spermidine or bis(ethy1)spermine (8890). These compoands exert a distinct antiproliferative action and can be used to control ornithine decarboxylase activity in DFMO resistant tumour cells (91). Bis(ethy1)spermine may be a useful inhibitor to control both ornithine decarboxylase and adenosylmethionine decarboxylase simultaneously (92). The achievement of a profound polyamine depletion by the inhibition of ornithine decarboxylase is a tough task as animal cells trigger a series of compensatory reactions in order to maintain sufficient intracellular polyamine pools. These include a secondary induction of adenosylmethionine decarboxylase (93, 94) and a striking accumulation of decarboxylated adenosylmethionine, which normally occurs only in trace amounts (95, 96). Putrescine and spermidine depletion also induce a dramatic enhancement of the uptake of exogenous polyamines and related compounds (97,98). Mouse (99,100) and human (101) tumour cells also rapidly acquire resistance to DFMO by overproducing ornithine decarboxylase owing to gene amplification. Human myeloma cells can acquire resistance to DFMO even without overproducing ornithine decarboxylase but instead enhancing arginase activity to such an extent that ornithine accumulated within the cell effectively competes with DFMO (102, 103).

Inhibition of Adenosylmethionine Decarboxylase The first inhibitor of any of the polyamine synthesizing enzymes to be discovered was methylglyoxal bis(guany1hydrazone) (MGBG, mitoguazone), an old anticancer drug Ann Med 23


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which appeared to be a potent inhibitor of adenosylmethionine decarboxylase (104). Although nearly 20 years have elapsed since the discovery of this compound as a polyamine antimetabolite, it still remains as probably the most important model compound for the design of inhibitors for adenosylmethionine decarboxylase. Although at first sight mitoguazone does not resemble the nucleoside S-adenosylmethionine, this compound nevertheless acts as a competitive inhibitor of adenosylmethionine decarboxylase (105, 106). MGBG lowers the concentrations of spermidine and spermine but provokes a striking accumulation of putrescine (68, 106). The massive accimulation of putrescine is not only based on the block of further utilisation but also on stimulation of ornithine decarboxylase activity (105, 107), probably through stabilisation of the enzyme (108, 109). Similarly, the powerful inhibition of diamine oxidase by MGBG, both in vitro (105) and in vivo (1l o ) , contributes to the accumulation of putrescine in some tissues. Unlike DFMO, which is remarkably specific to ornithine decarboxylase, MGBG exerts several effects that in all likelihood have nothing to do with the inhibition of adenosylmethionine decarboxylase or a fall in spermidine and spermine concentrations. These include, in addition to the above mentioned effects on ornithine decarboxylase and diamine oxidase, a profound antimitochondrial action seen as morphological as well as functional changes (1 11). The drug likewise affects lipid metabolism by inhibiting the carnitine dependent oxidation of long chain fatty acids (112). This inhibition of carnitine function may be directly related to the mitochondria1damage as the latter compound protects mitochondria from toxicity induced by MGBG (1 13). Irrespective of its exact mode of action, mitoguazone exerts clear cut antiproliferative effects on most animal cells at micromolar (1 to 10 pM) concentrations. Even though the inhibition of growth by mitoguazone can be reversed by adding exogenous polyamines, this phenomenon cannot be taken to mean that mitoguazone solely acts through an inhibition of spermidine and spermine accumulation, as the drug utilises the putative polyamine carrier for its cellular entry (1 14-1 16). Mammalian cells concentrate mitoguazone from the extracellular space remarkably effectively so that up to 1000-fold concentration gradients across the cell membrane are rapidly formed (116-1 17). The compound enters the cell by using the putative polyamine carrier and consequently polyamine depletion, which activates the carrier function, also strikingly enhances the cellular uptake of mitoguazone (97). MGBG works well in cultured cells and provoking a definite reduction in the concentrations of spermidine and spermine. The use of the compound in vivo is complicated by the inhibition of diamine oxidase, making it impossible to achieve any polyamine depletion in certain tissues displaying high diamine oxidase activity, such as rodent thymus (105). Of practical importance is the fact that animal, including human (118), intestinal lining has a high diamine oxidase activity aimed, in all likelihood, at preventing bacterial diamines and polyamines from entering the general circulation. When used as a chemotherapeutic agent in experimental mouse cancer models, MGBG almost totally blocks intestinal diamine oxidase activity (1 19) and strikingly promotes the entry of intestinal diamines into the systemic circulation (120). This inhibition of intestinal diamine oxidase has a great practical impact. This is especially so when mitoguazone is combined with inhibitiors of ornithine

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decarboxylase for example, DFMO in therapeutic regimens, since the intestine derived diamines, accumulating in the absence of diamine oxidase activity, finally reach the tumour cells and reverse the DFMO induced polyamine deprivation (120). The unwanted effects exerted by MGBG started a search, which is still in progress, for more suitable inhibitors of adenosylmethionine decarboxylase. The key issue in this search was to maintain powerful inhibition of adenosylmethionine decarboxylase but to abolish the inhibition of diamine oxidase. One solution was an alkylation of the molecule that appeared to result in a better affinity for the decarboxylase but a reduction in activity against diamine oxidase (121). The most potent derivative of MGBG was an alkylated compound ethylmethylglyoxal bis(guany1hydrazone) or EMGBG having a Ki value for adenosylmethionine decarboxylase less than 20 nM (122), yet inhibiting diamine oxidase decidedly less than does the parent compound. The alkylation of the glyoxal portion appeared to lead, however, to a loss of antiproliferative effects. In the primary screening studies MGBG and glyoxal bis(guany1hydrazone) (GBG), but not the more alkylated ethylglyoxal bis(guany1hydrazone)(EGBG), were listed as antileukaemic agents (123). When studying the relation between the structure and function of these bis(guanylhydrazones), Elo and coworkers (1 24) perceptively observed that the antiproliferative and antimitochondrial activity correlated with the portion of the compounds occurring as free base at physiological pH values - i.e., the portion of free base among the alkylated derivatives was much less than with GBG and MGBG. Hibasami and coworkers (125) described a new inhibitor of adenosylmethionine decarboxylase in which the guanyl groups of MGBG were replaced by dibutylamidino groups. This compound, methylglyoxal bis(buty1amidinohydrazone) (MGBB), inhibited adenosylmethionine decarboxylase with an apparent Ki value of 12 pM, a figure that is far above the inhibition constant of MGBG. interestingly, the compound also inhibited ornithine decarboxylase and spermidine synthase (125), depressed the growth of human erythroleukaemia cells (126) and prevented mouse skin tumour promotion (127). The latter group has further developed methylglyoxal bis(cyclopenty1amidino-hydrazone) as a multienzyme inhibitor of polyamine biosynthetic pathway (128). This compound likewise displays antileukaemic activity (128). Kramer and coworkers (129) introduced an irreversible inhibitor for adenosylmethionine decarboxylase. This compound, S-(5'-deoxy-5'-adenosylmethylthioethylhydroxylamine) (AMA) reduced spermidine and spermine concentrations by more than 80 %, while increasing putrescine content by a factor of 10 in cultured mouse L1210 leukaemia cells. With the combination of DFMO and AMA. Kramer et al were able to deplete the leukeamia cells of all three polyamines and showed that only spermidine (but not putrescine or spermine) fully supported the growth of L1210 cells during total polyamine deprivation (129).

Inhibitors of Spermidine and Spermine Synthases Nearly all experimental work with polyamine antimetabolites has been directed towards designing inhibitors of ornithine and adenosylmethionine decarboxylases, whereas relatively little attention has been paid to spermidine and spermine synthases. This strategy has apparently been


Polyamines: From Molecular Biology to Clinical Applications based on the following considerations: (i) Spermidine and spermine synthases occur in great excess compared with the two decarboxylases; (ii) A distinct growth inhibition can be achieved by depleting putrescine and spermidine (with the aid of inhibitors of ornithine decarboxylase) at normal or even at raised spermine levels; (iii) There is little need for a speficific inhibition of spermidine synthase as virtually complete spermidine depletion can be accomplished with the use of inhibitors of ornithine decarboxylase; (iv) As mentioned above, spermidine, and not putrescine or spermine, is the preferable polyamine to support cell proliferation (129). A more philosophical approach may be adopted by asking: What is the function of spermine in animal cells? It is not needed by most microorganisms, which contain only putrescine and spermidine and not spermine. In animal cells sperrnine may just be a salvage compound which can be converted back to spermidine and putrescine when needed. In any event, some activity has been directed to designing inhibitors for spermidine and spermine synthases. Methylthioadenosine (MTA), the by-product cleaved during the synthesis of spermidine and spermine, acts as a natural inhibitor of spermine synthase (130). This compound lowers the concentration of spermine and inhibits growth, in a way which is not, however, reversed by the addition of exogenous polyamines (130). Its alkylated derivative, namely S-methyl-5'-methylthioadenosine,produces a profound spermine depletion (and some increase in spermidine) with no growth inhibition (131). Similarly, N-alkylated derivatives of 1,3-diaminopropane appeared to be potent and specific inhibitors of spermine synthase (132). When added into rat hepatoma cell cultures, N-butyl-l,3-diaminopropane markedly decreased the concentration of spermine (with some increase in spermidine content) but did not affect the growth of the cells (132). These two studies likewise support the view that spermine may not be essential for animal cell proliferation. The most extensively studied inhibitors of spermidine synthase are cyclohexylamine (133) and S-adenosyl-l,8diamino-3-thiooctane (AdoDATO) (134), the latter being a transition-state analog of the transferase reaction. This compound has been tested in combination with DFMO resulting in a depletion of all three polyamines (including spermine) (135). It is, however, difficult to judge the contribution of spermine depletion to the antiproliferative effect exerted by the combination as DFMO alone produced almost total growth inhibition under these consitions (135). AdoDATO has also been reported to inhibit growth and differentiation of murine erythroleukemia cells (136). It appears that spermidine and spermine synthases are not meaningful targets for cancer chemotherapy, although they are interesting from enzymological point of view and possibly also helpful in elucidating the physiological function of the individual polyarnines.

Biosynthetic Enzymes of the Polyamines as Targets for Cancer Chemotherapy Polyamine antimetabolites have been widely used in several different animal tumour models and also in a few

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clinical trials during the past 10 years. As seen below, the initial experiments with tumour bearing animals seemed to offer real promise for future clinical use, yet these expectations have not been fullfilled. The concept is viable but the means (polyamine antimetabolites) are probably not ideal.

Use of Polyamine Anfimetabolites in Experimental Cancer Models The use of polyamine antimetabolites in experimental cancer is limited to the inhibitors of ornithine decarboxylase (DFMO in the great majority of cases) and to the bis(guanylhydroze) inhibitors of adenosylmethionine decarboxylase (mainly MGBG and EGBG). The irreversible inhibitors of ornithine decarboxylase have shown an impressive activity against several solid experimental cancers (such as prostatic, breast and pancreatic cancer), especially at low tumour burden (137-145). The compounds likewise are active against experimental leukaemias (such as L1210, P388 and T cell leukemia) (70, 141,146,147), although the increase in animal life span may not be that impressive. Even though the action of DFMO is only cytostatic and not cytocidal in the most experimental tumour models, this drug is remarkably effective, especially at low tumour burden. Inhibitors of ornithine decarboxylase effectively prevent metastases (148, 149) and inhibit tumour induced angiogenesis (150). DFMO can be conveniently administered in drinking water (2 Yo to 4 )."/ and is exceptionally well tolerated even during long term (21 year) treatment (151). The acute toxicities of DFMO are usually transient and not life threatening. These include interference in the formation of blood cell elements in the normal rat and suppression of the recovery of bone marrow hypoplasia induced by cytotoxic drugs (152). Rapidly proliferating tissues, such as intestinal lining, appear to be especially sensitive to the inhibition of polyamine biosynthesis (153). This drug, however, seems to have a specific side effect, first observed among cancer patients attending clinical trials with DFMO. The drug produced cochlear damage and a subsequent reversible hearing loss on long term use in human and experimental animals (154). In contrast to the irreversible inhibitors of ornithine decarboxylase, which show distinct activity against solid tumours, almost all the tumours sensitive to MGBG (the most widely used inhibitor of adenosylmethionine decarboxylase) are experimental leukaemias or lymphomas (123, 155). Already the early data generated more than 20 years ago showed that, although MGBG possessed antileukaemic activity fully comparable to those exerted by antimetabolites such as pyrimidine and purine analogs, the therapeutic range of the compound was extremely narrow: the ratio of LD, to LD,, was only 1.3 (the ratio for most anticancer drugs is over 2) (155). From among the congeners of MGBG, the parent compound GBG appears to possess an antileukaemic activity roughly comparable to that of MGBG (156, 157). The animal toxicity of MGBG include antiproliferative toxicity, cardiotoxicity, hepatotoxicity and irreversible hypoglycemia in rodent and rabbits (155, 158). In rodents the drug is rapidly excreted with no signs of cumulative toxicity (155, 159). As seen later, the animal toxicity pattern has limited value in predicting human toxicity. Alhonen-Hongisto et al. (97) showed that the depletion of putrescine and spermidine achieved with inhibitors of

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ornithine decarboxylade led to a strikingly enhanced accumulation not only of the natural polyamines but also of MGBG. The discovery laid the basis for the sequential combination of DFMO and MGBG which was subsequently tested, not only in experimental cancer models, but also in clinical practice. Studies with healthy mice showed that prior treatment with DFMO enhanced the accumulation of subsequently administered MGBG in selected tissues with high proliferative activity such as small intestine and bone marrow cells (159). These studies did not find any cumulative accumulation of MGBG on mouse tissues (160). Subsequent studies on mice inoculated either with Ehrlich ascites carcinoma cells (161) or with L1210 leukaemia cells (160) were even more promising, suggesting that pretreatment with DFMO selectively enhances the uptake of MGBG in the tumour cells, although this may not be true under all experimental conditions (162). A series of experimental therapeutic studies were subsequently carried out by employing a sequential or simultaneous combination of DFMO (or other irreversible inhibitors of ornithine decarboxylase) and MGBG. Combined treatment with DFMO and MGBG resulted in a synergistic anticancer effect in experimental mouse leukaemia (70, 141, 146, 160, 163, 164), in rat prostate cancer (139, 140, 165), in mouse renal adenocarcinoma (166) and in Lewis lung carcinoma (164). In some of these studies (139, 140) the combination of DFMO and MGBG seemed to reduce the toxicity of the latter drug. There are, however, contrasting examples of tumours that are sensitive to inhibitors of ornithine decarboxylase (mainly DFMO) but in which the sensitivity is lost when the drug is combined with MGBG (137,141). This phenomenon - i.e., the disapperance of DFMO induced cytostasis in drug regimens containing MGBG - has a clearly defined biochemical basis. The strong inhibition of diamine oxidase by MGBG (or its congeners), especially the almost total depression of the high enzyme activity residing in the intestinal epithelium (119), leads to an accelerated entry of intestine derived (dietary or bacterial) diamines or polyamines into the general circulation and ultimately into the tumour cells (IZO), and hence DFMO induced growth inhibition is lost. The latter phenomenon naturally implies that the tumours are sensitive to DFMO induced polyamine depletion but insensitive to MGBG, as is the case with most experimental solid tumours (155). In cases of rapidly growing leukaemias and lymphomas, which in general are sensitive to MGBG as a single agent, DFMO is actually used as a vehicle to enhance the cellular accumulation of MGBG, and the antiproliferative effect is exclusively based on the cytotoxicity of MGBG. Attempts have been made to circumvent the inhibition of intestinal diamine oxidase by MGBG (or its congeners) by using a polyamine free diet (164) or by using drug regimens also containing inhibitors of polyamine oxidase (167, 168).

Modulation of the Action of Conventional Cancer Chemotherapeutic Agents and Biological Response Modifiers by Polyamine Depletion Inhibitors of polyamine biosynthesis, most notably DFMO, have been combined with a variety of antineoplastic drugs with very different modes of action. As this topic has been covered in depth in recent book (169) only the major approaches and recent developments will be described Ann Med 23

here. As the mode of action of the polyamine antimetabolites is entirely different from that of the convential cancer chemotherapeutic agents, it is likely that in almost all cases inhibitors of polyamine biosynthesis potentiate other modes of cancer therapy. With a few exceptions, which seem to be limited to certain special cell types, the latter view is correct. In most cases the potentiating effect is simply additive, yet in some cell types, such as in Burkitt's lymphoma cells, DFMO acts synergistically with doxorubicin and cisplatin (170). Polyamine depletion achieved with inhibitors of ornithine decarboxylase enhances the radiosensitivity (171, 172) and thermosensitivity (173) of various tumour cells. In most cell types DFMO potentiates the antiturnour effect of compounds like cisplatin, various alkylating agents, mitomycin C, antimetabolites, interferons and antiestrogens (72, 174-181). An interesting exception to the general rule indicating that polyamine antimetabolites potentiate the antitumour efficacy of most convential anticancer drugs and physical treatments seems to be the rat brain tumour cell line (9L). Polyamine depletion achieved with the use of DFMO lessens the antitumour effect of aziridinylbenzoquinone (182) as well as that of vincristine and methotrexate (183), cisplatin (184) and Ara-C (185). A similar antagonistic action exerted by DFMO in combination with doxorubicin and chloranbucil has been reported to occur also in some human tumour cell lines (186, 187). In some cases the observed antagonism between polyamine antimetabolites and anticancer drugs, such as cisplatin, may be dependent on the schedule, as reported by Chang et al. (188). These authors found that DFMO given before cisplatin exerted an antagonistic action while its administration afterwards resulted in the polyamine antimetabolite acting synergistically with the platinium compound (188). So it seems that more systematic work is needed to optimise the design of combinations of polyamine antimetabolites with conventional cancer chemotherapeutic agents.

Experimental Chemoprevention of Cancer The use of inhibitors of polyamine biosynthesis as chemopreventive agents in individuals at high risk for cancer is an attractive approach. Inhibitors like DFMO appear to be especially suitable for the chemical prevention of cancer. This compound is relatively non-toxic, it works best at low tumour burden, and, most importantly, impressive experimental evidence exists showing that the compound indeed works in various experimental carcinogenesis models. DFMO and compounds alike apparently belong to the suppressive agents, according to the classification by Wattenberg (189), as they would prevent the occurrence of cancer in response to carcinogens. The experimental animal models in which DFMO has been employed as a chemopreventive agent include the two stage mouse skin carcinogenesis, chemically induced mammary carcinoma in rats, carcinogen induced tumours of the colon in mice and a rat bladder carcinoma model. The most widely studied animal model is promoter induced formation of papillomas in mouse skin. DFMO, given topically, parenterally or in drinking water, strikingly reduced the incidence of skin tumours and reduced the number of animals with tumours (190-1 92). Interestingly, in some of the experiments cited the dose of DFMO was substantially lower than those commonly used in the treatment of tumour bearing animals



(192). Nitrosourea induced mammary carcinogenesis in mice has served as another model to test the significance of polyamine biosynthesis for chemical carcinogenesis. A small amount (1 )."/ of DFMO in the drinking water dramatically cut the incidence of mammary tumours and resulted in a marked prolongation of cancer free time (193). As long term treatment with 1 Yo DFMO in drinking water apparently had some deleterious effects on growing females, the same authors extended their work to assessing the dose and found that as little as 0.125 Yo DFMO in drinking water significantly protected the animals from developing mammary tumours (194). Chemoprevention of tumours of the colon has similarly been achieved with DFMO alone (195) or with DFMO combined with the non-steroidal anti-inflammatory drug piroxicam (196). DFMO has also turned out to be effective in preventing urinary bladder carcinogenesis in the rat

(197, 198). There are apparent clinical applications for the use of DFMO as a chemopreventive agent, and it has been proposed to use DFMO in clinical practice to prevent recurrences of urinary bladder cancer (197) as well as for the chemoprevention of adenocarcinoma of the individuals at high risk for colonic polyposis. The fact that DFMO shows activity in the chemoprevention of cancer at a dose level I / 20 of that used for the treatment of experimental cancer also makes this drug attractive for clinical trials.

Clinical Experience with the Inhibitors of Polyarnine Biosynthesis as Anticancer Agents Although DFMO and its more potent congener, methyl acetylenic putrescine (MAP), have given promising results in the treatment of a variety of experimental tumors, including xenografted human neoplasms, their efficacy in the treatment of human cancer patients has not been accurately predicted by these studies. Some of the reasons for this partial failure are obvious. In the treatment of tumorbearing animals, the most common way of administering DFMO is to give the drug in drinking water as a 2 to 4 % solution. In terms of daily fluid intake this means a dose of 5 g/kg per day. It is obvious that a dose of this magnitude for man is beyond any practical considerations. Pharmacokinetic studies have shown that DFMO is slowly absorbed from the gastrointestinal tract with a bioavailability of about 60 Yo and that the plasma half-life appears to be substantially longer in man than in the mouse (199). Only a few, mainly phase I , clinical studies, have been carried out by employing inhibitors of ornithine decarboxylase (DFMO or MAP) as single cancer chemotherapeutic agents. These single agent studies include a phase I1 trial with DFMO administered orally to patients with advanced small cell lung cancer and previously untreated patients with metastatic colon cancer (200). Only minor therapeutic responses were observed among the small cell lung cancer patients. The toxicity pattern of DFMO is rather unique. Although gastrointestinal side effects are common in connection with oral treatment, they probably are only signs of some sort of osmotic diarrhea produced by massive oral doses of the drug. No signs of gastrointestinal toxicity occur during intravenous administration (201). Thrombocytopenia is rather common. Ototoxicity, however, appears to be specifically connected to long-term DFMO treatment

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irrespective of the route of administration (200). DFMO has also been given as a continuous intravenous infusion. Even though these studies (201)do not contribute much to the therapeutic profile of DFMO as a single agent, an interesting observation with clinical significance was made by Haegele et al. (202). They observed that an enhanced urinary excretion of decarboxylated adenosylmethionine, but not of the polyamines, is an excellent biochemical indicator of an effective inhibition of ornithine decarboxylase in humans (202). DFMO has likewise been administered through the hepatic artery in patients with liver metastases (203). Among nine patients in whom evaluation was possible, two obtained an objective partial response. Although ototoxicity frequently occurred, interestingly, there were no signs of thrombocytopenia or hepatic toxicity. This may be an approach worth further exploration. The putrescine derivative MAP has been used as a single agents in a phase I study. Nine patients with advanced malignancies received different doses of MAP (204). Although objective responses were not obtained, stabilisation of the disease was observed in about half of the patients. The fact that the inhibitor worked was confirmed by measuring the levels of urinary decarboxylated adenosylmethionine (204). Despite the mechanism of action of DFMO and MAP being the same - i.e., both are irreversible inhibitors of ornithine decarboxylase - there seems to be a marked difference between the toxicity of these drugs. For instance, MAP was not ototoxic, but there were signs of myelosuppression and renal toxicity (204). Based on this rather scarce clinical documentation, it seems that none of the currently known inhibitors of ornithine decarboxylase will prove the breakthrough for a single agent chemotherapeutic drug against cancer. Most of the clinical activity with DFMO has been carried out using combination regimens, most notably with MGBG. Based on our initial observation showing that DFMO enhances the uptake of subsequently administered MGBG, we designed a pharmacokinetic study using a sequential combination of the two inhibitors in childhood leukaemia (205). Prior treatment (peroral) with DFMO definitely enhanced the accumulation of subsequently infused MGBG in leukaemic blast cells and led to an apparent therapeutic synergism. This was shown by a rapid disappearance of the peripheral blast cells (1 16, 205). Toleration by patients of this combination was quite good and there were no signs that DFMO would enhance the toxicity of MGBG. In contrast to later studies with the same combination, intracellular (mononuclear leykocytes) MGBG levels were monitored and the dose of the latter drug adjusted accordingly (1 16,

205). The results of some of the subsequent clinical trials raised the possibility that the combination of DFMO and MGBG would merely enhance the side effects of MGBG or even produce unique unwanted effects without any real therapeutic advantage. This was the case in a trial consisting of patients with advanced solid tumours. Out of 28 evaluable patients only two had partial remission and the side effects in general were severe (206). Given the tumour types that were sensitive to MGBG, i.e. leukaemias and lymphomas but not the solid tumours, great success should not be expected with this combination. Warrell and his coworkers (207) carried out a trial with the sequential combination of DFMO and MGBG in patients with advanced cancer. Their experience was also negative: there was an apparent enhancement toxicity of MGBG with minimal therapeutic


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effect. The latter trial comprised 12 patients with lymphomas, which, according to the authors' previous experience (207), respond to single agent MGBG treatment. The combination, however, produced no observable objective responses (207). More encouraging were the preliminary results reported by Auclerc et al. (208), who obtained a 17 Yoobjective response rate in patients with various metastatic tumours. Maddox and her coworkers have done a series of phase Itrials with the sequential combination of DFMO and MGBG in patients with leukaemia (209-21 1). Although partial responses were achieved, the authors considered their results less impressive than those obtained by us (116, 205). They also felt that prior treatment with DFMO may enhance the toxicity of the subsequently administered MGBG, at least in their adult patients (209). The primary finding that DFMO increases the uptake of MGBG into peripheral mononuclear cells (205) was likewise confirmed (209). These authors (209-21 1) pointed out that the primary DFMO treatment must be sufficiently prolonged to allow polyamine depletion to develop before MGBG is administered. A further interesting observation was made: the combined treatment with the two polyamine antimetabolites seemed to promote white cell maturation in many patients (211). Gastaut et al. (212) treated 10 patients with acute myeloid leukaemia or blastic transformation with sequential combination of DFMO (6 g/m2/d) and intravenous MGBG (500 mgim') once a week. Although one complete response and four partial responses were obtained, this seemingly occurred at the cost of pronounced side effects. As the four patients with partial response were all undergoing the blast crisis of chronic myeloid leukaemia, the authors continued the trial by enrolling a further 10 patients, all at the blastic transformation stage of chronic myeloid leukaemia. The doses of both DFMO (4 g/m2/d) and MGBG (200 mg/mz weekly) were reduced. Complete or partial responses were obtained in four patients, with an acceptable level of toxicity (212). In addition to some anecdotal reports concerning the use of the DFMO/MGBG combination in prostate cancer (165) and in brain tumour patients (213, 214), the results of a recent phase 1-11 clinical trial (215) show that the combination displays impressive activity in patients with recurrent brain tumours. This finding is somewhat unexpected, as rodent studies (160) have shown that MGBG, given alone or in combination with DFMO, does not easily penetrate the blood brain barrier. This may not, however, be the case in human brain tumours, which are not protected by blood brain barrier (216). Based on the experimental work with the combination of DFMO and interferon (179, 217, 218), these studies were subsequently extended to phase I study in human patients with metastatic melanoma (219). Even though some objective responses were obtained, the therapeutic results of this trial were not particularly impressive, yet they probably warranted new studies. Although the clinical data available on the combined DFMOIMGBG regimen remain inconclusive, it seems that the approach is viable. Further studies on proper dosing and scheduling are badly needed. Some molecular manipulation of MGBG is likewise required to generate a more suitable inhibitor of adenosylmethionine decarboxylase to be used in combination with DFMO or any other inhibitor of ornithine decarboxylase. DFMO or its congeners are unique

drugs in many respects: their mode of action is entirely different from those of other anticancer drugs, as antiproliferative agents they are exceptionally well tolerated and the pattern of side effects is different from that of the conventional cancer chemotherapeutic agents. Unlike DFMO, the antileukaemic activity of MGBG was discovered much earlier, in 1958, than the inhibition of polyamine biosynthesis (156). The early experimental work was rapidly extended to clinical trials, especially in acute leukaemia (220-222). As the early clinical studies with MGBG have recently been reviewed in depth (223-225), only a brief summary is given here. Daily administration of MGBG produced striking responses in acute myelocytic leukaemia, complete responses ranging from 25 o/o to 69 '10 (223, 224). The responses were, however, obtained at the cost of severe toxicity, which included potential fatal effects such as severe mucotoxicity , gastrointestinal toxicity and refractory, possibly fatal, hypoglycemia (226, 227). Since these early studies the interest in the compound seemed to evaporate, although MGBG was sporadically used, mostly in combination regimens during the 1960s and 1970s (223, 224). During the early clinical work MGBG was administered as daily infusions, although in a few studies weekly or twice weekly administration was employed (228). Interest in this compound was revived when the Southwest Oncology Group in the United States introduced a once weekly infusion schedule in 1979 (229). This study showed that the intermittent administration improved the patients' toleration of the drug, apparently without reducing its anticancer activity (229). The re-evaluation of MGBG using a weekly or even biweekly administration schedule has been continued, but still the drug profile and toleration is arguable. Admittedly the new administration schedule has reduced the toxicity of the drug to an "acceptable level", but the new problem is its efficacy. MGBG as a single agent seems to have no or only marginal activity against most solid tumours (230-236). A small number of human neoplasms, such as lymphomas, appear to respond to monotherapy with or MGBG or multidrug regimens containing MGBG with acceptable toxicity (224, 225, 237, 238). MGBG seems to have established its status as a component in multidrug regimens. The best results have been obtained against lymphomas using a drug combination called MIME (MGBG, ifosfamid, methotrexate,etoposid) (225, 239-241 ). This combination has a curative potential Of 40 o /' to 60 /o' (241). What are the conclusions on the clinical usefulness of inhibitors of ornithine decarboxylase and of MGBG and its derivatives? Firm conclusions are probably premature at present. Although currently available polyamine antimetabolites are not breakthrough drugs in cancer chemotherapy, the biosynthetic enzymes of the polyamines remain and will remain as meaningful targets in the treatment of human cancer. Moreover, the real potential of the existing compounds has not been fully exploited. The first reliable method to monitor clinically the action of DFMO or any inhibitors of ornithine decarboxylase was introduced only a few years ago (202). Simple methods also exist (117, 205, 242) to monitor the cellular retention of MGBG and thus to predict the cumulative toxicity of the drug, but these methods have not gained widespread use in clinical studies. The fact remains that more chemical work has to be carried out to generate polyamine antimetabolites more suitable for clinical use than are DFMO and MGBG.


Potential Use of Polyamine Antimetabolites in Diseases Other than Cancer Although the chemotherapeutic approach has been by far the most important for developing polyamine antimetabolites, less systematic studies have been done on several other therapeutic approaches. These include hyperproliferative skin diseases, most notably psoriasis, certain infectious and parasitic diseases and the use of inhibitors of polyamine biosynthesis as contragestational agents.


Hyperproliferative Skin Diseases Hyperproliferative skin diseases have been natural targets for the chemical intervention of polyamine biosynthesis. Ultraviolet irradiation of hairless mouse skin not only induces epidermal proliferation, seen as enhanced DNA synthesis but also strikingly stimulates polyamine formation through an induction of ornithine decarboxylase (243246). This finding has made this animal model widely used in studies aimed at inhibiting epidermal proliferation through chemoprevention of polyamine biosynthesis. Although the initial studies by Seiler and Knodgen (244) showed that the inhibition of ornithine decarboxylase activity by DFMO after an exposure to ultraviolet radiation did not inhibit the stimulation of epidermal DNA synthesis, subsequent experiments showed that DFMO also moderately inhibited epidermal macromolecule synthesis (247-

250). After the establishment of the sequential administration schedule for DFMO and MGBG in cell cultures and in tumour bearing animal models, the same treatment regimen was applied to the prevention of epidermal proliferation in mouse skin (247, 249, 251, 252) and in cultured keratinocytes obtained from normal and psoriatic human skin (253). In each case the combination given either sequentially or concomitantly resulted in at least additive antiproliferative action as shown by inhibition of epidermal DNA and protein synthesis. A rescue concept was also developed for patients with epidermal MGBG levels that were too high (254). Unlike some tumour bearing animal models there were no signs of antagonism between DFMO and MGBG in this skin proliferation model. This probably means that epidermal tissue or skin in general is devoid of any significant diamine oxidase activity. Clinical experience with polyamine antimetabolites in the treatment of human psoriasis is almost non existent. Two small scale clinical trials with topical DFMO have been published, and in these DFMO produced epidermal polyamine depletion, but the clinical improvement was marginal (255, 256). Direct intradermal injection of DFMO into psoriatic lesions was shown to result in polyamine depletion (257). The overall progress in this field has generally been very slow or absent. The main difficulty in clinical work with the polyamine antimetabolites has been the polarity of the compounds and hence their poor penetration into the skin. As with cancer, doctors are looking for more suitable chemical entities to be used in the treatment of hyperproliferative skin diseases.

Infectious Diseases The currently available antimetabolites of polyamines have not turned out to be breakthrough drugs in cancer chemo-

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therapy, yet the synthesis of DFMO represents a milestone in the development of antiprotozoal agents. Polyamine antimetabolites may have potential as antiviral and antibacterial drugs, but in the case of antibacterial activity is complicated because unlike mammals bacteria have several alternative routes for the synthesis of the polyamines. As this question has recently been reviewed (87, 258), the reader is asked to consult these articles for further information. We will, however, briefly discuss the development of DFMO as an antiprotozoal agent. Although showing promise in several therapeutic areas, DFMO now seems to have found a group of diseases for which it really is a therapeutic breakthrough. These are parasitic protozoal infections, most notably those caused by trypanosomes affecting the health of millions of people in Africa and South America. The development of DFMO as an antiprotozoal agent began in the early 1980s, when Bacchi and his coworkers (259-261) found that the drug cured mice infected with Trypanosoma brucei brucei. From the start it was obvious that the curative effect of DFMO on trypanosomiasis was a result of polyamine depletion as the therapeutic effect could be blocked by a concomitant administration of putrescine or spermidine (261). Similar dramatic curative effects were observed when mice were infected with the human pathogens T. brucei rhodesiense or T. brucei gambiense and the cattle pathogen T. brucei congolense

(262, 263). Further extension of these studies showed than a combination of the anticancer antibiotic, bleomycin, with DFMO resulted in a therapeutic synergism, and this combination turned out to be highly effective in infections that affected the central nervous system (263-265). It seemed that even this combination worked through polyamine depletion, as the curative effect exerted by the combination in experimental trypanosomiasis was abolished by a concomitant administration of the polyamines (264). In addition to inhibitors of ornithine decarboxylase, inhibitors of adenosylmethionine decarboxylase may also be effective in experimental trypanosomiasis, as suggested by a recent report. An irreversible inhibitor of the latter enzyme ( a structural analog of decarboxylated adenosylmethionine) was shown to cure mice infected with either T. brucei brucei or T. brucei rhodesiense (266). Interestingly, treatment with this inhibitor resulted in spermidine depletion in the parasites associated with an increase in putrescine (266). In other words, it seems to be spermidine and not putrescine that is essential to the parasite [these organisms do not contain any spermine

(260)l.

Although the exact mechanism of action exerted by polyamine antimetaboliteson the trypanosome is not known, inhibition of nucleic acid synthesis and distinct morphological changes are associated with the depletion of putrescine and spermidine (263, 265). As in mammalian cells, the action of DFMO is cytostatic rather that cytocidal (267), and for the antitrypanosomal action by DFMO, an intact immunosystem of the host seems to be essential (268). What makes the trypanosomes so sensitive to polyamine deprivation? There are reports suggesting that trypanosomal ornithine decarboxylase has a much higher affinity for DFMO than does the mammalian enzyme (269). On the other hand, a fundamental difference exists between trypanosomal and mammalian ornithine decarboxylases, ornithine decarboxylase from the former lacking sequences


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at its carboxyl terminus which makes it completely stable in comparison with the mammalian enzyme (270, 271). In addition, trypanosomes are actively proliferating organisms and hence, like rapidly dividing mammalian cells, are sensitive to polyamine depletion. Furthermore, these creatures lack spermine, which in mammalian cells commonly increases in response to DFMO treatment. As to the clinical antiprotozoal activity of DFMO, the results are very promising, with the initial clinical trials already showing that DFMO exerts a dramatic and rapid therapeutic effects in patients with African sleeping sickness (268). The very first case reports were not entirely fortuitous, as ongoing clinical trials showed that the drug not only eradicated blood stream trypanosomes but also cured patients with late stage central nervous system involvement, a condition that so far has defied treatment (272). The treatment with DFMO also seems to be well tolerated, diarrhoea and anaemia being the main side effects (272). It seems, therefore, that antiprotozoal activity achieved through polyamine depletion is an effective new approach to cure trypanosomiasis. Some pilot experiments have been carried out in order to investigate whether polyamine deprivation would also be a meaningful approach to antimalarial therapy. DFMO decreased parasite growth in human red cells infected with malaria parasites (273) and inhibited macromolecular synthesis in the parasites (274). DFMO has a cytostatic effect on Plasmodium fakiparum as well as on trypanosomes (275). DFMO in combination with bis(benzy1)polyamine analogs is reported to cure murine malaria (276). Whether these experimental results will lead to clinical applications remains to be seen. Pneumocystis carinii pneumonia appears to be another protozoal infection sensitive to DFMO. The drug is effective in about half of the cases (277,278) thus clearly warranting further studies.

Polyamine Antimetabolites as Contragestational Agents Ornithine decarboxylase seems to play a critical role during early embryogenesis. Inhibition of ornithine decarboxylase activity and putrescine formation results in a 100 contragestational effect in mouse, rat and rabbit (279281). Providing DFMO in drinking water for murine females during days 5-9 (279, 280) and for pregnant rabbits during days 6-1 0 (281) effectively interrupts pregnancy. In rodents, DFMO has no effect on the outcome of pregnancy when administered during days 1-3 (282). Considering the low toxicity of DFMO and the short period of administration, it is interesting to note that apparently no efforts have been made to develop a new approach for postcoital contraception in man.

Miscellaneous Diseases The fact that inhibitors of ornithine decarboxylase act as antiproliferative agents and, among other properties, inhibit lymphocyte proliferation and immunoglobulin production (283) apparently prompted the idea that these compounds could be used as immunosuppressive agents to treat certain hyperimmune conditions. MAP (284) and DFMO (285) have indeed shown activity against lupus like disease in mice (MLR-lpr/lpr). Many of the pathological changes were counteracted by inhibitors of ornithine decarboxylase

in this experimental model for systemic lupus erythematosus (284, 285). MAP appears to be active in preventing collagen induced arthritis in mice through its immunosuppressive effects (286). In addition to the antiproliferative effects against lymphocytes, polyamine depletion induced prevention of Z-DNA formation could be responsible for the beneficial effects in lupus (284).

Concluding Remarks The aim of this article has not been to list all the relevant literature. The reader may have noticed that the extensive amount of literature covering the clinical chemistry polyamines has been omitted. One reason was because little real progress has been made to show directly that changes of the levels of extracellular polyamines could be specifically linked to some particular pathological condition in man. Admittedly, there may be some conditions in which the determination of extracellular polyamine concentrations may be of value. The molecular biology of the natural polyamines is a fascinating sector of basic research, and the secrets of the molecular genetics of ornithine decarboxylase, a unique mammalian enzyme, are only now being opened up. Even thousands of papers have appeared we still cannot unambiguously answer the question: What do the polyamines do? We hope, however, that sufficient experimental evidence has been presented to justify the view that undisturbed formation of the polyamines is a prerequisite for animal cell proliferation to occur. As to the clinical applications of polyamine research, it looks as though the initial enthusiasm of the early 1980s has to some extent been tempered. This is mainly because the very promising experimental work performed with various cancer bearing animal models could not be directly translated into clinical practice. Clinical treatments for psoriasis or hyperproliferative skin diseases have to be awaited. On the other hand, polyamine antimetabolites have proved a real breakthrough in the treatment of certain parasitic diseases, most notably African trypanosomiasis. A summary of the clinical status of polyamine antimetabolites could read as follows: the approach is viable, but the compounds are not yet ready. We gratefully acknowledge the skilful secretarial assistance of Ms. Taru Koponen.

References 1. Mann T, Lutwak-Mann C. Male reproductive function and semen. Berlin, Heidelberg,New York: Springer-Verlag1981: 1-495. 2. Russell D, Snyder SH. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat

liver, chick embryo, and various tumors. Proc Natl Acad Sci USA 1968; 60: 1420-7. 3. Pegg AE, Williams-Ashman HG.Biosynthesisof putrescine in the prostate gland of the rat. Biochem J 1968; 108: 533-

9. 4. Janne J, Raina A. Stimulationof spermidine synthesis in the regenerating rat liver: relation to increased ornithine decarboxylase activity. Acta Chem Scand 1968; 22: 134951. 5. Russell DH, Snyder SH. Amine synthesis in regeneratingrat liver: extremely rapid turnover of ornithine decarboxylase. Mol Pharmacol 1969; 5: 253-62. 6. Hayashi S-l (ed.). Ornithine decarboxylase: Biology,

Polyarnines: From Molecular Biology to Clinical Applications

7. 8.

9. 10.


11.

12. 13.

14.

15. 16. 17.

enzymology, and molecular genetics. New York: Pergamon Press, 1989: 1-147. McConlogue L, Gupta M, Wu L, Coffino P. Molecular cloning and expression of the mouse ornithine decarboxylase gene. Proc Natl Acad Sci USA 1984; 81 : 540-4. Kontula KK, Torkkeli TK, Bardin CW, Janne OA. Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied bv comolementarv DNA. Proc Natl Acad Sci USA 1984; 81: 7i1-5.' Kahana C. Nathans D. Isolation of cloned cDNA encodina mammalian ornithine decarboxylase. Proc Natl Acad S6 USA 1984; 81 : 3645-9. Pegg AE. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J 1986; 234: 249-62. Janne J, Williams-Ashman HG. On the purification of Lornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme. J Biol Chem 1971 ; 246: 172532. Rogers S,Wells R, Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 1986; 234: 364-8. Ghoda L, van Daalen Wetters T, Macrae M, Ascherman D, Coffino P. Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 1989; 243: 1493-5. Hickok NJ, Seppanen PJ, Gunsalus GL, Janne OA. Complete amino acid sequence of human ornithine decarboxylase deduced from complementary DNA. DNA 1987; 6: 179-87. Coffino P,Chen EL.Nucleotidesequenceof mouseornithine decarboxylase gene. Nucleic Acids Res 1988; 16: 2731. Eisenberg LM, Janne OA. Nucleotide sequence of the 5'flanking region of the murine ornithine decarboxylase gene. Nucleic Acids Res 1989; 17: 2359. Wen L, Huang J-K, Blackshear PJ. Rat ornithine decarboxylase. Nucleotide sequence, potential regulatory elements. and cornoarison the the mouse aene. J Biol Chem 1989; 264 901 6-21. van Steea H. van Oostrom TM. Hodemaekers HM, van Kreyl CF.-Cloning and functional analysis of the rat ornlthine decarboxylase-encoding gene. Gene 1990; 93: 249-56. van Steeg H, van Oostrom CThM, Martens JWM, van Kreyl CF, Schepens J, Wieringa B. Nucleotide sequence of the human ornithine decarboxylase gene. Nucleic Acids Res 1989; 17: 8855-6. Fitzgerald MC, Flanagan MA. Characterization and sequence analysis of the human ornithine decarboxylase gene. DNA 1989; 8: 623-34. Moshier JA, Gilbert JD, Skunca M, Dosescu J, Almodovar KM, Luk GD. Isolation and expression of a human ornithine decarboxylase gene. J Biol Chem 1990; 265: 4884-92. Hickok NJ, Wahlfors J, Crozat A, Halmekyto M, Alhonen L, Janne J, Janne OA. Human ornithine decarboxylase-encoding loci: nucleotide sequence of the expressed gene and characterization of a pseudogene. Gene 1990; 93: 257-63. Winqvist R, Makela TP, Seppanen P, Janne OA, AlhonenHongisto L, Janne J, Grzeschik K-H, Alitalo K. Human ornithine decarboxylase sequences map to chromosome regions 2pter p23 and 7cen qter but are not amplified with the NMYC oncogene. Cytogenet Cell Genet 1986; 42: 133-40. Cox, DR, Trouillo T, Ashley PL, Brabant M, Coffino P. A functional mouse ornithine decarboxylase gene (Odc) maps to chromosome 12: further evidence of homology between mouse chromosome 12 and the short arm of human chromosome 2. Cytogenet Cell Genet 1988; 48: 9 2 4 . Srinivasan PR,Tonin PN, Wensing EJ, Lewis WH.Thegene for ornithine decarboxylase is co-amplified in hydroxyurearesistant hamster cells. J Biol Chem 1987; 262: 12871-8. Tonin PN, Yeger H, Stallings RL, Srinivasan PR, Lewis WH. Amplification of N-myc and ornithine decarboxylase genes in human neuroblastoma and hydroxyurea-resistant hamster cell lines. Oncogene 1989; 4: 11 17-21. Janne J, Poso H, Raina A. Polyamines in rapid growth and cancer. Biochim Biophys Acta 1978; 473: 241-93. Pajunen A, Crozat A, Janne OA, lhalainen R, Laitinen PH, Stanley B, Madhubala R, Pegg AE. Structure and regulation of mammalian S-adenosylmethionine decarboxylase. J Biol Chem 1988; 263: 17040-9. Pegg AE, Wiest L, Pajunen A. Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts of rat

30. 31. 32.

33.

34. 35. 36. 37. 38. 39. 40.

I

18. 19.

20. 21. 22.

23.

24.

25. 26.

27. 28.

29.

41.

42.

43. 44. 45. 46. 47.

48.

49.

50.

51,

52.

253

prostate. Biochem Biophys Res Commun 1988; 150: 78893. Pegg AE. Evidence for the presence of pyruvate in rat liver Sadenosylmethionine decarboxylase. FEBS Lett 1977; 84: 33-6. Stanley BA, Pegg AE, Holm I. Site of pyruvate formation and processing of mammalian S-adenosylmethionine decarboxylase proenzyme. J Biol Chem 1989; 264: 21073-9. Poso H, Hannonen P, Himberg JJ, Janne J. Adenosylmethionine decarboxylase from various organisms: relation of the putrescine activation of the enzyme to the ability of the organism to synthesize spermine. Biochem Biophys Res Commun 1976; 68: 227-34. Shirahata A, Takeshima T, Samejima K. Monospecific antiserum to rat spermidine synthase and its application to rat tissues and several mammals. J Biochem 1988; 104,71721. Wahlfors J, Alhonen L, Kauppinen L, Hyvonen 1,Janne J, Eloranta TO. Human spermidine synthase: cloning and primary structure. DNA Cell Biol 1990; 9: 103-1 0. Pinel JPJ, Gorzalka BB, Ladak F. Cadaverine and Dutrescine initiate the burial of dead conspecifics by rats. Physiol Behav 1981; 27: 81 9-24. Kahana C, Nathans D. Translational regulation of mammalian ornithine decarboxylase by polyamines. J Biol Chem 1985; 260: 15390-3. Holtta E, Pohjanpelto P. Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. J Biol Chem 1986; 261 9502-8. Persson L, Holm I, Heby 0. Translational regulation of ornithine decarboxylase by polyamines. FEBS Lett 1986; 205: 175-8. Kameji T, Pegg AE. Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines. J Biol Chem 1987; 262: 2427-30. Grens A, Scheffler IE. The 5'- and 3'-untranslatedregionsof ornithine decarboxylase mRNA affect the translational efficiency. J Biol Chem 1990; 265: 11810-6. Ito K, Kashiwagi K, Watanabe S,Kameji T, Hayashi S, lgarashi K. Influence of the 5'-untranslatedregion of ornithine decarboxylase mRNA and spermidine on ornithine decarboxylase synthesis. J Biol Chem 1990; 265: 13036-41. van Daalen Wetters T, Macrae M, Brabant M, Sittler A, Coffino P. Polyamine-mediated regulation of mouse ornithine decarboxylase is posttranslational. Mol Cell Biol 1989; 9: 5484-90. Schimke RT, Doyle D. Control of enzyme levels in animal tissues. Annu Rev Biochem 1970; 39: 929-76. Tabor CW, Tabor H. 1,4-Diaminobutane (putrescine), spermidine, and spermine. Annu Rev Biochem 1976; 45: 285-306. Scalabrino G, Ferioli ME. Polyamines in mammalian tumors. Part I. Adv Cancer Res 1981; 35: 151-268. Scalabrino G, Ferioli ME. Polyamines in mammalian tumors. Part II. Adv. Cancer Res 1982; 36: 1-102. Alhonen-Hongisto L, Kallio A, Sinervirta R, Janne OA, Gahmberg CG, Janne J. Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells. Biochem J 1985; 229: 71 1-5. Polvinen K, Sinervirta R, Alhonen L, Janne J. Overproduction of ornithinedecarboxylase confers an apparent growth advantage to mouse tumor cells. Biochem Biophys Res Commun 1988; 155,373-8. Janne J, Alhonen L, Halmekyto M, Hyttinen J-M, Sinervirta R, Utriainen M, Myohanen S, Wahlfors J, Kiiskinen M, Voipio H-M. Production of transgenic mice carrying human and mouse ornithine decarboxylase gene constructs. Life Chemistry reports 1991 (in press). Doerfler W. The significance of DNA methylation patterns: promoter inhibition by sequence-specific methylation is one functional consequence. Philos Trans R SOC Lond [Biol] 1990; 326: 253-65. Ben-Hattar J, Beard P, Jiricny J. Cytosine methylation in CTF and Spl recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro. Nucleic Acids Res 1989; 17: 10179-90. Doerfler W, Toth M, Kochanek S, Achten S, FreisemRabien U, Behn-Krappa A, Orend G. Eukaryotic DNA methylation: facts and problems. FEBS Lett 1990; 268: 329-


254


33. 53. Weissbach A, Ward C, Bolden A. Eukaryotic DNA methylation and gene expression. Current Topics in Cell Regul 1989; 30: 1-21. 54. Selker EU. DNA methylation and chromatin structure: a view from below. TlBS 1990; 15: 103-7. 55. Meehan RR, Lewis JD, McKay S,Kleiner EL, Bird AP. lndentification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 1989; 58: 499507. 56. Antequera F, Macleod D, Bird AP. Specific protection of methylated CpGs in mammalian nuclei. Cell 1989; 58: 50917. 57. Swain JL, Stewart TA, Leder P. Parental legacy determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting. Cell 1987; 50: 719-27. 58. Riggs AD. DNA methylation and cell memory. Cell Biophys 1989; 15: 1-13. 59. Sapienza C, Paquette J, Tran TH, Peterson A. Epigenetic and genetic factors affect transgene methylation imprinting. Development 1989; 107: 165-8. 60. teinonen P, Alhonen-Hongisto L, Laine R, Janne OA, Janne J. Chronic exposure to dexamethasone induces hypomethylation of ornithine decarboxylase genes in a human myeloma cell line. FEBS Lett 1987; 215: 68-72. 61. Alhonen-Hongisto L, Leinonen P, Sinervirta R, Laine R, Winqvist R, Alitalo K, Janne OA, Janne J. Mouse and human ornithine decarboxylase genes: methylation polymorphism and amplification. Biochem J 1987; 242: 20510. 62. Lipsanen V, Leinonen P, Alhonen L, Janne J. Hypomethylation of ornithine decarboxylasegene and Erb-Aloncogene in human chronic lymphaticleukemia. Blood 1988; 72: 20424. 63. Halrnekyto M, Hirvonen A, Wahlfors J, Alhonen L, Janne J. Methylation of human ornithine decarboxylase gene before transfection abolishes its transient expression in Chinese hamster ovary cells. Biochem Biophys Res Commun 1989; 162: 528-34. 64. Pegg AE, Williams-Ashman HG. Stimulation of the decarboxylation of S-adenosylmethionine in mammalian tissues. Biochem Biophys Res Commun 1968; 30: 76-82. 65. Pegg AE, Williams-Ashman HG. On the role of S-adenosylmethionine in the biosynthesis of spermidine by rat prostate. J Biol Chem 1969; 244: 682-93. 66. Kameji T, Pegg AE. Effect of putrescine on the synthesis of S-adenosylmethionine decarboxylase. Biochem J 1987; 243: 285-8. 67. Metcalf BW, Bey P, Danzin C, Jung MJ, Casara J, Vevert JP. Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C.4.1.1.17) by substrate and product analogues. J Am Chem SOC1978; 100: 2551-3. 68. Heby 0, Janne J. Polyamine antimetabolites: Biochemistry, specificity and biological effects of inhibitors of polyamine synthesis. In: Morris DR, Marton LJ, eds. Polyamines in Biology and Medicine. New York: Marcel Dekker Inc. 1981: 243-3 10. 69. Luk GD, Goodwin G, Gazdar AF, Baylin SB. Growth-inhibitory effects of DL-alpha-difluoro-methvlornithine in the spectrum of human lung carcinoma cells ih culture. Cancer Res. 1982; 42; 3070-3. 70. Sunkara PS. Prakash NJ. Chana CC. Sioerdsma A. Cytotoxicity of methylglyoxal bis(guany1hydrazbne) in combination with alpha-difluoromethylornithineagainst HeLa cells and mouse L1210 leukemia. J Natl Cancer lnst 1983; 70: 505-9. 71. Pera PJ, Kramer DL, Sufrin JR, Porter CW. Comparison of the biological effects of four irreversible inhibitors of ornithine decarboxylase in two murine lymphocytic leukemia cell lines. Cancer Res 1986; 46: 1148-54. 72. Sunkara PS, Fowler SK, Nishioka K. Selective killing of transformed cells in combination with inhibitors of polyamine biosynthesis and S-phase specific drugs. Cell Biol Int Rep 1981; 5: 991-7. 73. Seidenfeld J, Block AL, Komar KA, Naujokas MF. Altered cell cycle phase distributions in cultured human carcinoma cells partially depleted of polyamines by treatment with difluoromethylornithine.Cancer Res 1986; 46: 47-53. 74. Schindler J, Kelly M, McCann PP. Inhibition of ornithine

75. 76.

77. 78.

79. 80. 81. 82. 83.

84. 85. 86. 87.

88.

89.

90.

91.

92.

93. 94.

95.

decarboxylase induces embryonal carcinoma cell differentiation. Biochem Biophys Res Commun 1983; l14: 41 0-7. Oredsson SM, Billgren M, Heby 0. Induction of F9 embryonal carcinomacell differentiationby inhibition of polyamine synthesis. Eur J Cell Biol 1985; 38: 335-43. Schindler J, Kelly M, McCann PP. The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alphadifluoromethylornithine.J Cell Physiol 1985; 122: 1-6. Kapyaho K, Janne J. Stimulation of melanotic expression in murine melanomacells exposedto polyamine antimetabolites. Biochem Biophys Res Commun 1983; 113: 18-23. Sunkara PS, Chang CC, Prakash NJ, Lachmann, PJ. Effect of inhibition of polyamine biosynthesis by DL-alphadifluoromethylornithine on the growth and melanogenesis of B16 melanoma in vitro and in vivo. Cancer Res 1985; 45: 4067-70. Kapyaho K, Sinervirta R, Janne J. Effects of inhibitors of polyamine biosynthesis on the growth and melanogenesis of rnurine melanoma cells. Cancer Res 1985; 45: 1444-8. Niskanen EO, Kallio A, McCann PP, Baker DG. The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice. Blood 1983; 61 : 740-5. Chen KY, Nau D, Liu AY-C. Effects of inhibitors of ornithine decarboxylase on the differentiation of mouse neuroblastoma cells. Cancer Res 1983; 43: 2812-8. Sugiura M, Shaftman T, Kufe D. Effects of polyamine depletion on proliferation and differentation of murine erythroleukemia cells. Cancer Res 1984; 44: 1 4 4 0 4 . Sugiura M, Shaftman T, Mitchell T, Griffin J, Kufe D. Involvement of spermidine in proliferation and differentiation of human promyeloticleukemiacells. Blood 1984;63: 11538. Ewton DZ, Erwin BG, Pegg AE, Florini JR. The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts. J Cell Physiol 1984; 120: 263-70. Bethell DR, Pegg AE. Polyamines are needed for the differentiation of 3T3-Ll fibroblasts into adipose cells. Biochem Biophys Res Commun 1981; 102: 272-8. Erwin BG, Bethell DR, Pegg AE. Role of polyamines in differentiation of 3T3-Ll fibroblasts into adipocytes. Am J Physiol 1984; 246: c293--c300. Janne J, Alhonen-Hongisto L. Inhibitors of ornithine decarboxylase: Biochemistry and applications. In: Hayashi S, ed. Ornithine decarboxylase: Biology, enzymology, and molecular genetics. New York: Pergamon Press, 1989: 59-85. Porter CW, Ganis B, Vinson T, Marton LJ, Kramer DL, Bergeron RJ. Comparison and characterization of growth inhibition in L1210 cells by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and N1 ,NB-bis- (ethyl)spermidine, an apparent regulator of the enzyme. Cancer Res 1986; 46: 6279-85. Porter CW, Berger FG, Pegg AE, Ganis 6, Bergeron RJ. Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine. Biochem J 1987; 242: 4 3 3 4 0 . Porter CW, Bergeron RJ. Enzyme regulation as an approach to interference with polyamine biosynthesis - an alternativeto enzyme inhibition. Adv Enzyme Regul1988; 27, 57-79. Pegg AE, Madhubala R, Karneji T, Bergeron J. Control of ornithine decarboxylase activity in alpha-difluoromethylornithine-resistant L1210 cells by polyamines and synthetic analogues. J Biol Chem 1988; 263: 11008-14. Porter CW, Pegg AE, Ganis B, Madhabala R, Bergeron RJ. Combined regulation of ornithine and S-adenosyl-methionine decarboxylase by spermine and the spermine analogue N1N12-bis(ethyl)spermine. Biochem J 1990; 268: 207-1 2. Alhonen-Hongisto L. Regulation of S-adenosylmethionine decarboxylase by polyamines in Ehrlich ascites-carcinoma cells grown in culture. Biochem J 1980; 190: 747-54. Marnont PS, Joder-Ohlenbusch A-M, Nussli M, Grove J. Indirect evidence for a strict negative control of S-adenosylL-methionine decarboxylase by spermidine in rat hepatoma cells. Biochem J 1981; 196: 41 1-22. Marnont PS, Danzin C, Wagner J, Siat M, JoderOhlenbusch A-M, Claverie N. Accumulation of decarboxylated S-adenosyl-L-methionine in mammalian cells as consequence of the inhibition of putrescine biosynthesis. Eur J Biochem 1982; 123: 499-504.


Polyamines: From Molecular Biology to Clinical Applications 96. Pegg AE, Poso H, Shuttlewoth K, Bennett RA. Effect of inhibition of polyamine synthesis on the content of decarboxylated S-adenosylmethionine.Biochem J 1982; 202: 519-26. 97. Alhonen-Hongisto L, Seppanen P, Janne J. lntracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guany1hydrazone).Biochem J 1980; 192: 941-5. 98. Wallace HM, Melvin MAL, Keir HM. Increased uptake of polyamines by baby-hamster kidney cells (BHK-21/C13) treatedwith alpha-methylornithine. Biochem SocTrans 1982; 10: 456. 99. Alhonen-Hongisto L, Kallio A, Sinervirta R, Seppanen P, Kontula KK, Janne OA, Janne J. Difluoromethylornithineinduced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells. Biochem Biophys Res Commun 1985; 126: 734-40. 100. Alhonen-Hongisto L, Hirvonen A, Sinervirta R, Janne J. Cadaverine supplementation during a chronic exposure to difluoromethylornithine allows an overexpression, but prevents gene amplification of ornithine decarboxylase in L1210 mouse leukaemia cells. Biochem J 1987; 247: 651-5. 101. Leinonen P, Alhonen-Hongisto L, Laine R, Janne OA, Janne J. Human myelomacells acquire resistance to difluoromethylornithine by amplification of ornithine decarboxylase gene. Biochem J 1987; 242: 199-203. 102. Alhonen-Hongisto L, Leinonen P, Laine R, Janne J. Human myeloma cells acquire resistance to difluoromethylornithine without overproducing ornithine decarboxylase. Biochem Biophys Res commun 1987; 144: 132-7. 103. Hirvonen A, Eloranta T, Hyvonen T, Alhonen L, Janne J. Characterization of difluoromethylornithine-resistant mouse and human cell lines. Biochem J 1989; 258: 709-1 3. 104. Williams-Ashman HG, Schenone A. Methylglyoxal bis(guanylhydrazone) as a potent inhibitor of mammalian and yeast S-adenosylmethioninedecarboxylase.Biochem Biophys Res Commun 1972; 46: 288-95. 105. Holtta E, Hannonen P, Pispa J, Janne J. Effect of methylglyoxal bis(guany1hydrazone)on polyamine metabolism in normal and regenerating rat liver and in rat thymus. Biochem J 1973; 136: 669-76. 106. Janne J, Alhonen-Hongisto L, Nikula P, Elo H. SAdenosylmethionine decarboxylase as target of chemotherapy. Adv Enzyme Regul 1986; 24: 125-39. 107. Heby 0, Sauter S, Russell DH. Stimulation of ornithine decarboxylaseactivity and inhibition of S-adenosyl-L-methione decarboxylase activity in leukemic mice by methyl-glyoxal bis(guany1-hydrazone).Biochem J 1973; 136: 1121-4. 108. Karvonen E, Poso H. Stabilizationof ornithine decarboxylase and N1-spermidine methyltransferase in rat liver by methvlalvoxal bis(auanv1hvdrazonel. Biochim Bioohvs Acta , , 19841761 : 239-43. 109. Nikula P. Alhonen-Honaisto L. Janne J. Effects of bis(gbanylnydrazones) on thgactlvity and expression of ornithine decarboxylase Biochem J 1985; 231.21 3-6 110 Pegg AE, McGill SM Inh bition of diamine oxidase by 1 , l (~methvlethane-divlidene)-oinitro)-bis-[3-aminoa~an1dine) and l’,l-((methylethanediylidjne)-dir;itrilo):diguani~ne. Biochem Pharmacol 1978; 27: 1625-9. 111. Pathak SN, Porter CW, Dave C. Morphological evidence for an antimitochondria action by methylglyoxal-bis(guany1hydrazone). Cancer Res 1977; 37: 2246-9. 112. Nikula P, Alhonen-Hongisto L, Seppanen, Janne J. Inhibition of long-chain fatty acid oxidation by methylglyoxal bis(guany1hydrazone).Biochem Biophys Res Commun 1984; 120: 9-1 4. 113. Nikula P, Ruohola H, Alhonen-Hongisto L, Janne J. Carnitine prevents the early mitochondria1damage produced by methylglyoxal bis(guany1hydrazone) in L1210 leukaemia cells. Biochem J 1985; 228: 513-6. 114. Dave C, Caballes L. Studies on the uptake of methylglyoxalbis(guanyihydraz0ne)(CH3-G)and spermidine (Spd)in mouse leukemia sensitive and resistant to CH3-G (L121O/CH3-G). Fed Proc 1973; 32: 736. 115. Seppanen P. Some properties of the polyamine deprivationinducible uptake system for methylglyoxal bis(guany1hydrazone) in tumor cells. Acta Chem Scand 1981; 835: 731-6. 116. Janne J, Alhonen-Hongisto L, Seppanen P, Siimes M. Use of polyamine antimetabolites in experimental tumors and

. ,

255

in human leukemia. Med Biol 1981; 59: 448-57. 117. Seppanen P, Alhonen-Hongisto L, Siimes M, Janne J. Quantitation of methvlalvoxal bis(auanv1hvdrazone)in blood plasma and leukemia cells of p a t h s ieceiving thedrug. Int J Cancer 1980; 26: 571-6. 118. Bieganski T, Kuscge J, Lorenz W, Hesterberg R, Stahlknecht C-D, Feussner K-D. Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism. Biochirn Biophys Acta 1983; 756: 196-203. 119. Kallio A, Janne J. Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites. Biochem J 1983; 21 2: 895-8. 120. Kallio A, Nikula P, Janne J. Transfer of intestine-derived diamines into turnourcellsduring treatment of Ehrlich ascitescarcioma-bearing mice with polyamine anti-metabolites. Biochem J 1984; 218: 641-4. 121. Janne J, Morris DR. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methyl-glyoxal bis(guany1hydrazone) and their cellular uptake during lymphocyte activation. Biochem J 1984; 218: 947-51. 122. Elo H, Mutikainen I, Alhonen-Hongisto L, Laine R, Janne J, Lumme P. Biochemical properties and crystal structure of ethylmethylglyoxal bis(guany1hydrazone) sulfate - an extremely powerful novel inhibitor of adenosylmethionine decarboxylase. 2 Naturforsch 1986; 41c: 851-5. 123. Mihich E. Bis-guanylhydrazones. Handbookof Experimental Pharmacology, New York: Springer Verlag, 1975; vol. 37: 766-88. 124. Elo H0,Tilus PTE, MutikainenIP, Riekkola M-L. Remarkable differences between the species distributions of various bis(guany1hydrazones)at physiological conditions, and their possible involvement in the strict structural requirements for antileukemic activity. Anti-Cancer Drug Design 1989;4: 3039. 125. Hibasami H, Tsukada T, Maekawa S, Nakashima K. Methylglyoxal bis(bisbuty1amidinohydrazone).a new inhibitor of polyamine biosynthesis that simultaneously inhibits ornithine decarboxylase, adenosylmethionine decarboxylase and spermidine synthase. Biochem Pharmacol 1986; 35: 2982-3. 126. Hibasami H, Tsukada T, Maekawa S, Nakashima K. Antitumor effect of methylglyoxal bis(butylarnidin0-hydrazone), a new inhibitor of S-adenosylmethionine decarboxylase, against human erythroid leukemia K 562 cells. Cancer Lett 1986; 30: 17-23. 127. HibasamiH,TsukadaT,MaekawaS,SukuraiM,Nakashima K. Inhibition of mouse skin tumor promotion and of promoterinduced epidermal polyamine biosynthesis by methylglyoxal bis(buty1amidinohydrazone).Carcinogenesis 1988; 9: 199202. 128. HibasamiH,MaekawaS, MurataT,NakashirnaK.Antitumor effect of a new multienzyme inhibitor of polyamine synthetic pathway, methylglyoxal-bis(cyclopentylamidinohydrazone)$ against human and mouse leukemia cells. Cancer Res 1989; 49: 2065-8. 129. Kramer DL, Khomutov RM, Bukin YV, Khomutov AR, Porter CW: Cellular characterization of a new irreversible inhibitor of S-adenosylmethionine decarboxylase and its use in determining the relative abilities of individual polyamines to sustain growth and viability of L1210 cells. Biochem J 1989; 259: 325-31. 130. Pajula R-L, Raina A. Methylthioadenosine, a potent inhibitor of spermine synthase from bovine brain. FEBS Lett 1979; 99: 343-5. 131. Pegg AE, Coward JK. Growth of mammalian cells in the absence of the accumulation of spermine. Biochem Biophys Res Commun 1985; 133: 82-9. 132. Baillon JG, Kolb M, Mamont PS. Inhibition of mammalian spermine synthase by N-alkylated-l,3-diaminopropanederivatives in vitro and in cultured rat hepatoma cells. Eur J Biochem 1989; 179: 17-21. 133. HibasamiH,Tanaka M, Nagai J, IkedaT. Dicyclohexylamine, a potent inhibitor of spermidine synthase in mammalian cells. FEBS Lett 1980; 116: 99-1 01. 134. Tang K-C, Pegg AE, Coward JK. Specific and potent inhibition of spermidine synthase by the transition-state analog, S-adenosyl-3-thio-1 &diaminooctane. Biochem Biophys Res Commun 1980; 96: 1371-7.

256


135. Pegg AE, Tang K-C, Coward JK. Effects of S-adenosyl-l,8diamino-3-tiooctane on Dolvaminemetabolism. Biochemistrv 1982; 21, 5082-9. 136. Sherman ML. SharfmanTD. Coward JK. Kufe DW. Selective inhibition of spermidine biosynthesis anddifferentiation by Sadenosyl-l,8-diamino-3-thiooctane in murine erythroleukemia cells. Biochem Pharmacol 1986; 35: 2633-6. 137. Prakash NK, Schechter PJ, Mamont PS, Grove J, KochWeser J, Sjoerdsma A. Inhibition of EMT6 tumor growth by interference with polyamine biosynthesis: effects of alphadifluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase. Life Sci 1980; 26: 181-94. 138. Dunzendorfer U. Therapie der Prostatakarzinoms mit Polyaminsynthese-inhibitoren. Urol Int 1982; 37: 349-57. 139. Dunzendorfer U, Releya NM, Kleinert E, Balis ME, Whitmore WF. Antigrowth effect of some inhibitors of polyamine synthesis on transplantable prostate cancer. Oncology 1983; 40: 57-62. 140. Herr HW, Kleinert EL. Antigrowth effect of polyamine biosynthesis inhibitors on the dunning R 3327-G prostatic tumor. Prostate 1984; 5: 439-44. 141, Bartholeyns J, Mamont P, Casara P. Antitumor properties of (2R, 5R)-6-heptyne-2,5-diamine, a new potent enzyme activated irreversible inhibitor of ornithine decarboxylase in rodents. Cancer Res 1984; 44: 4972-7. 142. Marx M, Townsend CM, Barranco SC, Glass EJ, Thompson JC. Treatment of hamster pancreatic cancer with adifluoromethylornithinean inhibitorof polyamine biosynthesis. J Natl Cancer lnst 1987; 79: 543-8. 143. Carvalho L, Foulkes K, Mickey DD. Effect of DMSO and DFMOon rat prostate tumor growth. Prostate 1989; 15: 12333. 144. Mickey DD, Carvalho L, Foulkes K: Conventional chemotherapeutic agents combined with DMSO or DFMO in treatment of rat prostate carcinoma. Prostate 1989; 15: 22132. 145. Manni A, Badger B, Glinkman P, Bartholomew M,Santner S, Demers L. Individual and combined effects of adifluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats. Cancer Res 1989; 49: 3529-34. 146. Bartholeyns J, Koch-Weser J. Effects of alpha-difluoromethylornithine alone and combined with adriamycin or vindesine on L1210 leukemia in mice, EMT6 solid tumors in mice, and solid tumors induced by injection of hepatoma tissue culture cells in rats. Cancer Res 1981 ; 41 : 5158-61. 147. Smart LM, MacLachlan G, Wallace HM, Thomson AW. Influence of cyclosporin a and a-difluoromethylornithine, an inhibitor of polyamine biosynthesis, on two rodent T-cell cancers in vivo. Int J Cancer 1989; 44: 1069-73. 148. Kubota S, Ohsawa N, Takaku F. Effects of DL-alphadifluoromethylornithine on the growth and metastasis of B16 melanoma in vivo. Int J Cancer 1987; 39: 244-7. 149. Sunkara PS, Rosenberger AL. Antimetastatic activity of DLalpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis in mice. Cancer Res 1987; 47: 933-5. 150. Takigawa M, Enomoto M,Nishida Y, Pan H-0, Kinshita A, Suzuki F. Tumor angiogenesis and polyamines: a-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits 6 16 melanoma-inducedangiogenesis in ovo and the proliferation of vascular endothelial cells in vitro. Cancer Res 1990; 50: 4131-8. 151. Luk GD, Abeloff MD, McCann PP, Sjoerdsma A, Baylin SB. Long-term maintenance therapy of established human small cell variant luna carcinoma imDlants in athvmic mice with a cyclic regimen of difluoromethylornithine. Cancer Res 1986; 46: 1849-53. 152. Slotkin TA, Seidler FJ, Trepanier PA, Whitmore WL, Lerea L, Earner GA, Weigel SJ, Bartolome J. Ornithine decarboxylase and polyamines in tissues of the nenatal rat: effects of alpha-difluoromethylornithine,a specific, irreversible inhibitor of ornithine decarboxylase. J Pharmacol Exp Ther 1982; 222: 741-5. 153. Yarrington JT, Sprinkle DJ, Loudy DE, Diekerna KA, McCann PP, Gibson JP. Intestinal changes caused by DLalpha-difluoromethylornithine (DFMO)an inhibitor of ornithine decarboxylase. Exp Mol Patholl983; 39: 300-16. 154. Salzer SJ, Mattox DE, Brownell WE. Cochleardamage and increased threshold in alpha-difluoromethylornithine (DFMO) treated guinea pigs. Hear Res 1990; 46: 101-12.


I

4--

&",.A n q

,

155. Mihich E. Methvlalvoxal-bis(auanv1hvdrazone).an aaent with activity in acute myelotic leul?emia and lymphomas.krch ltal Pat Clin Tumori 1965; 8: 153-206. 156. Freedlander BL, French FA. Carcinostatic action of polycarbonyl compounds and their derivatives. (I. Glyoxal bis(guany1hydrazone) and derivatives. Cancer Res 1958; 18: 360-3. 157. French FA, Freedlander BL, Hoskins A, French J. Carcinostatic activity of some dicarbonyl compounds and their bishydrazones. Acta Union Int Contra Cancrum 1960; 16: 614-24. 158. Porter CW, Dave C, Mihich E. Inhibition of S-adenosylmethionine decarboxylase as an approach in cancer therapeutics. In: Morris DR, Marton LJ, eds. Polyamines in Biology and Medicine. New York: Marcel Dekker, 1981: 407-36. 159. Kallio A, Seppanen P, Alhonen-Hongisto L, Janne J. Modulation of the tissue disposition of methylglyoxal bis(quanv1hvdrazone)in mice bv DolvaminedeDletion and bv polyamine administration. Cancer Res 1983; 43: 324-7. ' 160. Seppanen P. Alhonen-Hongisto L. Janne J. Combined use of 2:difluoromethylornithine-and methylglyoxal bis(guany1hydrazone) in normal and leukemia-bearing mice. Cancer Lett 1983; 18: 1-1 0. 161. Seppanen P, Alhonen-Hongisto L, Janne J. Polyamine deprivation-induced enhanced uptake of methylglyoxal bis(guany1hydrazone) by tumor cells. Biochim Biophys Acta 1981 ; 674: 169-77. 162. Kramer DL, Paul B, Porter CW. Effect of pretreatment with alpha-difluoromethylornithine on the selectivity of methylglyoxal bis(guany1hydrazone) for tumor tissue in L1210 leukemic mice. Cancer Res 1985; 45: 251 2-5. 163. Ask A, Persson L, Oredsson SM, Heby 0. Synergistic antileukemic effect of two polyamine synthesis inhibitors. Host survival and cell-cycle kinetic analysis. Int J Cancer 1986; 37: 465-70. 164. Nakaike S, Kashiwagi K, Terao K, Ito K, lgarashi K. Combined use of alpha-difluoromethylornithineand inhibitors of S-adenosylmethioninedecarboxylase in mice bearing P388 leukemia or Lewis lung carcinoma. Jpn Cancer Res 1988; 79: 501-8. 165. Dunzendorfer U, Knoner M. Therapie mit lnhibitoren der Polyaminbiosynthese bein refraktaren Prostatakarzinom. Onkologie 1985; 8: 196-200. 166. Herr HW, Kleinert EL, Conti PS, Burchenal JH, Whitmore Jr WF. Effects of alDha-difluoromethvlornithineand methvlglyoxal bis-(guanylhydrazone) on thegrowth of experimenial renal adenocarcinoma in mice. Cancer Res 1984; 44: 43825. 167. Sarhan S, Knodgen B, Seiler N.The gastrointestinal tract as polyarnine source for tumor growth. Anticancer Res 1989; 9: 215-24. 168. Seiler N, Sarhan S, Grauffel C, Jones I?, Knodgen B, Moulinoux J-P. Endogenous and exogenous polyamines in support of tumor growth. Cancer Res 1990; 50: 5077-83. 169. Porter CW, Janne J. Modulation of antineoplastic drug action by inhibitors of polyamine biosynthesis. 1987) In: McCann PP, Pegg AE, Sjoerdsma A, eds. Inhibition of polyamine metabolism: Biological significance and basis for new therapies. New York: Academic Press Inc, 1987: 203-48. 170. Allen ED, Natale RB. Effect of alpha-difluoromethylornithine alone and in combination with doxorubicin hydrochloride, cisdiamminechloroplatinum (II),and vinblastine sulfate on the growth of P3J cells in vitro. Cancer Res 1986; 46: 3550-5. 171. Arundel CM, Nishioka K, Tofilon PJ. Effects of alphadifluoromethylornithine-induced polyamine depletion on the radiosensitivity of a human colon carcinoma cell line. Radiat Res 1988; 114: 634-40. 172. Courdi A, Milano G, Bouclier M, Lalanne CM. Radiosensitization of human tumor cells by alpha-difluoromethylornithine. Int J Cancer 1986; 38: 103-7. 173. Fujimoto S, Shrestha RD, Ohta M, lgarashi K, Endoh F, KokubunM,KoikeS,TogawaY,OkuiK. Enhancedantitumor efficacy with a combination of hyperthermochemotherapy and thermosensitization with polyamine antimetabolites in nude mice. JDn J Suraerv 1987: 17: 1 10-7. 174. Chang BK, Black Jr Ihhibition of the growth of pancreatic cancer cell lines bv aloha-difluoromethvlornithine (DFMOI alone and combineb with cis-diaminediiydrochloroplatinum (DDP). Proc Am Ass Cancer Res 1983; 24: 271.

6.


Polyamines: From Molecular Biology to Clinical Applications 175. Cavanaugh Jr PF, Pavelic ZP, Porter CW. Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea- induced cytotoxicity and DNA damage by alpha-difluoromethylornithine in L1210 leukemia cells. Cancer Res 1984; 44: 3 8 5 6 6 1 . 176. Marton LJ, Levin VA, Hervatin SJ, Koch-Weser J, McCann PP, Sjoerdsma A. Potentiation of the antitumor therapeutic effects of 1,3-bis(2-chIoroethyl)-l-nitrosourea by alphadifluoromethylornithine, an ornithine decarboxylase inhibitor. Cancer Res 1981; 41 : 4426-31. 177. Seidenfeld J, Komar KA. Chemosensitization of cultured human carcinomacells to 1,3-bis(2-chloroethyl)-1 -nitrosourea by difluoromethylornithine-induced polyamine depletion. Cancer Res 1985; 45: 2132-8. 178. Fujimoto S, lgarashi K, Shrestha RD, Miyazaki M, Okui K. Antitumor effects of two polyamine antimetabolites combined with mitomycrn C on human stomach cancer cells xenotransplanted into nude mice. Int J Cancer 1985; 35: 821-5. 179. Sunkara PS, Prakash NJ, Mayer GD, Sjoerdsma A. Tumor suppression with a combination of alpha-difluoromethylornithine and interferon. Science 1983; 219: 851-3. 180. Bregman MD, Meyskens Jr FL. Difluoromethylornithine enhances inhibition of melanoma cell growth in soft agar by dexamethasone, clone a interferon and retinoic acid. Int J Cancer 1986; 37: 101-7. 181, Thomas T, Kiang DT. Additive growth-inhibitory effects of DL-alpha-difluoromethylornithine and antiestrogens on MCF7 breast cancer cell line. Biochem Biophys Res Commun 1987; 148: 1 3 3 8 4 5 . 182. Alhonen-Hongisto L, Deen DF, Marton LJ. Decreased cytotoxicity of aziridinylbenzoquinone caused by polyamine depletion in 9L rat brain tumor cells in vitro. Cancer Res 1984; 44: 3 9 4 2 . 183. Alhonen-Hongisto L, Hung DT, Deen DF, Marton LJ. Decreased cell kill of vincristine and rnethotrexate againts 9L rat brain tumor cells in vitro caused by alpha-difluoromethylornithine-induced polyamine depletion. Cancer Res 1984; 44: 4440-2. 184. Oredsson SM, Deen DF, Marton LJ. Decreased cytotoxicity of cis-diamminedichloroplatinum (11) by alpha-difluoromethylornithinedepletion of polyamines in 9L rat brain tumor cells in vitro. Cancer Res 1982; 42: 1296-9. 185. Oredsson SM, Gray JW, Deen DF, Marton LJ. Decreased cytotoxicity of 1-beta-D-arabino-furanosylcytosine in 9L rat brain tumor cells pretreated with alpha-difluoromethylornithine in vitro. Cancer Res 1983; 43: 2541-4. 186. Seidenfeld J, Komar KA, Naujokas MF, Block AL. Reduced cytocidal efficacy foradriamycin in cultured human carcinoma cells depleted of polyamines by difluoromethylornithine treatment. Cancer Res 1986; 46: 1155-9. 187. Seidenfeld J, Komar KA, Naujokas MR, Block AL. Effects of DFMO-induced polyamine depletion on human tumor cell sensitivity to antineoplastic DNA-cross-linking drugs. Cancer Chemother Pharmacol 1986; 17: 16-20. 188. Chang BK, Gutman R, Chou T-C. Schedule-dependent interaction of alpha-difluoromethylornithineand cis-diamminedichloroplatinum (11) against human and hamster pancreatic cancer cell lines. Cancer Res 1987; 47: 2247-50. 189. Wattenberg LW. Chemoprevention of cancer. Cancer Res 1985; 45: 1-8. 190. Takigawa M, Verm AK, Simsiman RC, Boutwell RK. Polyamine biosynthesis and skin tumor promotion: inhibition of 12-0-tetra-decanoylphorbol-13-acetate-promoted mouse skin tumor formation by the irreversible inhibitor of ornithine decarboxylase alpha-difluoromethylornithine. Biochem Biophys Res Commun 1982; 105: 969-76. 191. Weeks CE, Herrman AL, Nelson FR, Slaga TJ. Alphadifluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits tumor promoter-induced polyamine accumulation and carcinogenesis in mouse skin. Proc Natl Acad Sci USA 1982; 79: 6028-32. 192. Takigawa M, Verma AK, Simsiman RC, Boutwell RK. Inhibition of mouse skin tumor promotion and of promoterstimulatd epidermal polyamine biosynthesis by alphadifluoromethylornithine. Cancer Res 1983; 43: 3732-8. 193. Thompson HJ, Herbst EJ, Meeker LD, Ronan AM,Minocha R. Inhibition of 1 methylnitrosourea-induced mammary tumorigenesis by difluoromethylornithine. Carcinogenesis (Lond.) 1984; 5: 1649-51. 194. Thompson HJ, Meeker LD, Herbst EJ, Ronan AM, Minocha R. Effect of concentration of D,L-2-difluoromethylornithineon

195.

196.

197.

198.

199.

200.

201. 202.

203.

204.

205.

206. 207. 208.

209.

210.

21 1.

21 2.

257

murine mammary carcinogenesis. Cancer Res 1985; 45: 1170-3. Kingsnorth AN, King WWK, Diekema KA, McCann PP, Ross JS, Malt RA. Inhibition of ornithine decarboxylase with 2-difluoromethylornithine: reduced incidence of dimethvlhvdrazine-induced colon tumors in mice. Cancer Res 1683,43: 2545-9. Reddv BS. Navini J. Tokumo K. Riaottv J. Zana E. Kelloff G. Chemoprevention of colon carci6ge;esis byconcurrent administration of piroxicam, a nonsteroidai anti-inflammatory drug with D,L-a-difluoromethylornithine, an ornithine decarboxylase inhibitor, in diet. Cancer Res 1990; 50: 25628. Homma Y, Ozono S,Numata I, Seidenfeld J, Oyasu R. Inhibition of carcinogenesis by alpha-difluoromethylornithine in heterotopically transplanted rat urinary bladders. Cancer Res 1985; 45: 648-52. Nowels K, Homma Y, Seidenfeld J, Oyasu R. Prevention of inhibition effects of alpha-difluoromethylornithine on rat urinary bladder carcinogenesis by exogenous putrescine. Cancer Biochem Biophys 1986; 8: 2 5 7 - 6 3 . Haegele KD, Alken RG, Grove J, Schechter PJ, KochWeser J. Kinetics of alpha-difluoromethylornithine: an irreversible inhibitor of ornithine decarboxvlase. Clin Pharmacol Ther 1981; 30: 210-7. Abeloff MD, Rosen ST. Luk GD. Bavlin SB. Zeltman M. Sjoerdsma A. Phase II trials of alpha-difiuoromethylornithine; an inhibitor of polyarnine synthesis, in advanced small cell lung cancer and colon cancer. Cancer Treat Rep 1986; 70: 843-5. Maddox AM, Keating MJ, McCredie KE, Estey E, Freireich EJ. Phase I evaluation of intravenous difluoromethylornithine - a potent inhibitor. Invest New Drugs 1985; 3: 287-92. Haegele KD, Splinter TAW, Romijn JC, Schechter PJ, Sjoerdsma A. Decarboxylated-S-adenosyimethionine excretion: a biochemical marker of ornithine decarboxylase inhibition by alpha-difluoromethylornithine. Cancer Res 1987; 47: 890-5. Lipton A, Harvey HA, Glenn J, Weidner WA, Strauss M, MillerSE,Taylor JB, White-Hershey D, Barlow LR.Aphase I study of hepatic arterial infusion using difluoromethylornithine. Cancer 1989; 63: 433-7. Cornbleet MA, Kingsnorth A, Tell GP, Haegele KD, JoderOhlenbusch A-M. Phase I study of methylacetylenic putrescine, an inhibitor of polyamine biosynthesis. Cancer Chemother Pharmacol 1989; 23: 348-52. Siimes M, Seppanen P, Alhonen-Hongisto L, Janne J. Synergistic action of two polyamine antimetabolites leads to a raDid theraoeutic resDonse in childhood leukemia. Int J Canker 1981 28: 5 6 7 l 7 0 . W i n t e r TA. Romiiin JC. Phase I studv of abha-difluoromethylornithke and methyl-GAG. Eur J 'Cancer Clin Oncol 1986; 22: 61-7. Warrell Jr RP, Coonley CJ, Burchenal JH. Sequential inhibitionof polyamine synthesis. Cancer Chemother Pharmacol 1983; 11: 134-6. Auclerc M-F, Weil M, Jacquillat CI. Preliminary clinical results of the combination of methylglyoxal guanylhydrazone (M.GAG) and difluoromethylornithine (DFMO). Proc Am SOC Clin Oncol 1983; 2: 43. Maddox A-M, Freireich EJ, Keating MJ, Haddox MK. Alterations in bone marrow and blood mononuclear cell polyamine and methylglyoxal bis(guany1hydrazone) levels: Phase I evaluation of alpha- difluoromethylornithine and bis(guany1hydrazone) treatment of human hematological malignancies. Cancer Res 1988; 48: 1367-73. Maddox A-M, Keating MJ, Freireich EJ, Haddox MK. Polyamines increase in human peripheral blood and bone marrow mononuclear cells following administration of methylglyoxal bis(guany1hydrazone). Chemotherapy 1988; 34: 419-29. Maddox A-M, Keating MJ, Freireich EJ, Haddox MK. Mononuclear cell polyamine content associated with myeloid maturation in patients with leukemia during administration of polyamine inhibitors. Invest New Drugs 1989; 7: 119-29. Gastaut J-A, Tell G, Schechter PJ, Maraninchi D, Mascret B, Carcassonne Y. Treatment of acute myeloid leukemia and blastic phase of chronic myeloid leukemia with combined eflornithine(alpha-difluoromethylornithine)and methylglyoxalbis- guanylhydrazone (methyl-GAG). Cancer Chemother

258


Pharmacol 1987; 20: 344-8. 213. Wilson CB, Gutin PH. New therapeutic approaches for brain tumors. Cancer 1984; 54: 2702-5. 214. Levin VA. Chemotherapy of primary brain tumors. Neurol Clin 1985; 3: 855-66. 21 5. Levin VA, Chamberlain MC, Prados MD, Choucair AK,

216.


217.

218.

219.

220. 221.

222. 223.

224. 225.

226. 227. 228. 229. 230. 231. 232. 233. 234. 235.

236.

Berger MS, Silver P, Seager M, Gutin PH, Davis RL, Wilson CB. Phase 1-11 study of eflornithine and mitoguazone combined in the treatment of recurrent primary brain tumors. Cancer Treat Rev 1987; 71 : 459-64. Rosenblum MG, Stewart DJ, Yap BS, Leavens M, Benjamin RS, Loo TL. Penetration of methylglyoxal bis(guany1hydrazone) into intracerebral tumors in humans. Cancer Res 1981; 41: 459-62. Sunkara PS, Prakash NJ, Rosenberger AL, Hagan AC, Lachmann PJ, Mayer GG. Potentiation of antitumor and antimetastatic activities of alpha-difluoromethylornithine by interferon inducers. Cancer Res 1984; 44: 2799-802. Heston WDW, Fleischmann J, Tackett RE, Ratliff TL. Effects of alpha-difluoromethylornithine and recombinant interferon-alpha2 on the growth of a human renal cell adenocarcinoma xenograft in nude mice. Cancer Res 1984; 44: 3220-5. Talpaz M, Plager C, Quesada J, Benjamin R, Kantarjian J, Gutterman J. Difluoromethylornithine and leukocyte interferon: a DhaSe I studv in cancer oatients. Eur J Cancer Clin Oncol 1986; 22: 6851-9. Freireich EJ. Frei 111 E. Karon M. Methvlalvoxal bis(auanv1hydrazone): a new agent active againkf acute myelocybc leukemia. Cancer Chemother Rep 1962; 16: 183-6. Levin RH, Brittin GM, Freireich EJ. Different patterns of remission in acute myelocytic leukemia. A comparison of the effects of methyl-glyoxal-bis-guanylhydrazone and 6mercaptopurine. Blood 1963: 21 : 689-97. Levin RH, Henderson E, Karon M, Freireich EJ. Treatment of acute leukemia with methylglyoxal-bis-guanylhydrazone (methyl GAG). Clin Pharmacol Ther 1964; 6: 3 1 4 2 . Janne J, Holtta E, Kallio A, Kapyaho K. Role of polyamines and their antimetabolites in clinical medicine. In: Cohen MP, Foa PP, eds. Special Topics in Endocrinology and Metabolism, New York: Alan R Liss Inc, 1983; vol 5: 227-93. Warrell Jr RP, Burchenal JH. Methylglyoxal-bis(guany1hydrazone) (Methyl-GAG):current status and future prospects. J Clin Oncol 1983; 1 : 52-65. Hoffmann H, Gutsche W, Amlacher R, Schulze W, Werner W, Lenk H, Wohlrab W, Haupt E.Mitoguazon (Methylglyoxalbis guanylhydrazon): Stand und Perspektiven. Arch Geschwulstforsch 1989; 59: 1 3 5 4 8 . Mihich E. Current studies with methylglyoxal bis(guany1hydrazone). Cancer Res 1963; 23: 1375-89. Regelson W, Holland JF. Initial clinical study of parenteral methylglyoxal bis(guany1hydrazone) diacetate. Cancer Chemother Rev 1961: 11: 81-6. Shnider BI, Colsky J, Jones R, Carbone PP. Effectiveness 01 methyl-GAG (NSC-32946)administered intramuscularlv Cancer Chemother Rev 1974; 58: 689-95. Knight 111 WA, Livingston RB, Fabian C, Costanzi J. Phase 1-11 trial of methyl-GAG: A Southwest oncology group pilot study. Cancer Treat Rev 1979; 63: 1933-7. Scher H, Chapman R, Kelsen D, Gralla R, Wittes R. Phase II trial of mitoguazone in patients with relapsed small cell carcinoma of the lung. Cancer Treat Rev 1984; 68: 561-2. Vogl SE, Pagano M, Horton J. Phase II study of methylglyoxal bis-guanylhydrazone (NSV 3296) in advanced ovarian cancer. Am J Clin Oncol 1984; 7: 733-6. Chun H, Bosl GJ. Phase II trial of rnitoguazone in patients with refractory germ cell tumors. Cancer Treat Rev 69; 4612. Sordillo PP, Magill GB, Welt S. Phase II trial of methylglyoxalbis-guanylhydrazone (methyl-GAG) in patients with soft-tissue sarcomas. Am J Clin Oncol 1985; 8: 316-8. Thongprasert B, Bosl GJ, Geller NL, Wittes RE. Phase II trial of rnitoguazone in patients with advanced head and neck cancer. Cancer Treat Rep 1984; 68: 1301-2. lnamasu MS, Oishi N, Chen TT, Kraut EH, Grozea PN, Constanzi JJ, Bonnet JD. Phase I1study of mitoguazone in pancreatic cancer: a Southwest Oncology Group Study. Cancer Treat Rev 1986; 70: 531-2. Moore MR, Graham SD, Birch R, Irwin L. Phase II evaluation of mitoguazone in metastatic hormone-resistant prostate

cancer: a Southeastern cancer study group trial. Cancer Treat Rev 1987; 71 : 89-90. 237. Dana BW, Jones SE,Coltman C, Stuckey WJ. Salvage treatment of unfavourable non-Hondgkin’s lymphoma with cisplatin, amsacrine, and mitoguazone: a Southwest Oncology Group Pilot Study. Cancer Treat Rev 1986; 70: 291-2. 238. Warrell RP, Danieu L, Coonley CJ, Atkins C. Salvage chemotherapy of advanced lymphoma with investigational drugs: mitoguazone, gallium nitrate, and etoposide. Cancer Treat Rev 1987; 71 : 47-51. 239. Cabanillas F, Hagemeister FB, McLaughlin P, Velasquez WS, Riggs S, Fuller L, Smith T. Results of MIME salvage regimen for recurrent or refractory lymphoma. J Clin Oncol 1987; 5: 407-12. 240. Hagemeister FB, Tannir N, McLaughlin P, Salvador P, Riggs S, Velasquez WS, Cabanillas F. MIME chemotherapy (Methyl-GAG, ifosfamide, methotrexate, etoposide) as treatment for recurrent Hodgkin’s disease. J Clin Oncol 1987; 5 : 556-61. 241. Cabanillas F, Velasquez WS, McLaughin P, Jagannath S, Hagemeister FB, Redman JR, Swan F, Rodriquez MA. Results of recent salvage chemotherapy regimens for lymphoma and Hodgkin’s disease. Semin Hematol1988; 25: 47-50. 242. Seppanen P, Alhonen-Hongisto L, Janne J, Raina A, Eloranta T. Indirect determination of methylglyoxal bis(guanylhydrazone) in leukocytes of leukemia patients. Clin Chim Acta 1982; 123: 187-92. 243. Lowe N, Verma AK, Boutwell RK. Ultraviolet light induces epidermal ornithine decarboxylase activity. J Invest Dermatol 1978; 71: 417-0. 244. Seiler N, Knodgen B. Effects of ultraviolet light on epidermal polyamine metabolism. Biochem Med 1979; 21 : 168-81. 245. Mizuno N, Kono T, Tanii T, lshii M, Hamasa T, Yoshida H, Otani S, Morisawa S. Increase in the expression of the ornithine decarboxylase gene in mouse skin by ultraviolet light. Arch Dermatol Res 1989; 281: 514-6. 246. Hillebrand GG, Winslow MS, Benzinger MJ, Heitmeyer DA, Bissett DL. Acute and chronic ultraviolet radiation induction of epidermalornithinedecarboxylase activity in hairless mice. Cancer Res 1990; 50: 1 5 8 0 4 . 247. Kapyaho K, Lauharanta J, Janne J. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination. J Invest Dermatol 1983; 81: 102-6. 248. Lowe NJ. Epidermal ornithine decarboxylase, polyamines, cell proliferation and tumor promotion. Arch Dermatol 1980; 116: 822-5. 249. McCullough JL, Pechkam P, Klein J, Weinstein GD, Jenkins JJ. Regulation of epidermal proliferation in mouse epidermis by combination of difluoromethyl ornithine (DFMO) and methylglyoxal bis(guany1hydrazone) (MGBG). J Invest Dermatol 1985: 85: 518-21. 250 Eshbaugh WG Jr, Forley BG, Ritter EF,Serafin D, Klitzman B. Stimulation of DNA synthesis in human ep dermis bv UVB radiation and its inhibition by difluoromethylornithine. Plastic Reconstr Surg 1990; 85: 593-6. 251. Kapyaho K, Linnamaa K, Janne J. Effect of epidermal polyamine depletion on the accumulation of methylglyoxal bis(guany1hydrazone) in mouse skin. J Invest Dermatoll982; 78: 3 9 1 4 . 252. Kapyaho K, Lauharanta J, Janne J. Combined topical use of difluoromethyl ornithine and methylglyoxal bis(guany1hydrazone) in UV-irradiated mouse skin. Br J Dermatol1982; 107: 41 5-22. 253. Kapyaho K, Kariniemi A-L, Virtanen I,Janne, J. Effect of polyamine antimetabolites on cultured human keratinocytes from normal and uninvolved psoriatic skin. Br J Dermatol 1984; 1111403-11. 254. Janne, J. Alhonen-Hongisto L, Kapyaho K, Seppanen P. Experimental approaches to the systemic and topical use of polyamine antimetabolites as antiproliferative agents. Adv Polyamine Res 1983; 4: 17-32. 255. Grosshans E, Henry M, Tell G, Schechter P, Bohlen P, Grove J, Koch-Weser J. Les polyamines dans le psoriasis. Ann Dermatol Venereol (Paris) 1980; 107: 377-87. 256. Henry M, Juillard J,Tell G, Schechter PJS, Grove J, KochWeser J, Grosshans E. Comparative effects of topical anthralin and difluoromethylornithine (alphaDFM0) on epidermal polyamine concentration in psoriasis. Br J Dermatol


Polyamines: From Molecular Biology to Clinical Applications 1981; 105: supplement 20,33-4. 257. Kousa M, Kapyaho K, Lauharanta J, Linnamaa K, Janne J, Mustakallio K. Methylglyoxal bis(guany1hydrazone) and alpha- difluoromethylornithine-inducedpolyamine deprivation in psoriatic lesions. Acta Derm Venereol 1982; 62: 2 2 1 - 4 . 258. Tyms AS, Williamson JD, Bacchi CJ. Polyamine inhibitors in antimicrobial chemotherapy. J AntimicrobChemother 1988; 22: 403-27. 259. BacchiCJ, Nathan HC, Hutner SH, McCann PP,Sjoerdsma A. Polyamine metabolism: A potential therapeutic target in Trypanosomes. Science 1980; 210: 3 3 2 4 . 260. Bacchi CJ. Content, synthesis, and function of polyamines in Trypanosomatids: relationship to chemotherapy. J Protozool 1981; 28: 20-7. 261. Nathan HC, Bacchi CJ, Hutner SH, Rescigno D,McCann PP, Sjoerdsma A. Antagonism by polyamines of the curative effects of alpha-difluoromethylornithinein Trypanosoma brucei infections. Biochem Pharmacol 1981; 30: 3010-3. 262. Karbe E, Bottger M, McCann PP, Sjoerdsma A, Freites EK. Curative effect of alpha-difluoromethylornithine on fatal Trypanosoma congolence infection in mice. Tropenmed Parasit 1982; 33: 161-2. 263. Bacchi CJ, McCann PP, Nathan HC, Hutner SH,Sjoerdsma A. Antagonism of polyamine metabolism - a critical factor in chemotheraov of African Trvoanosomiasis. Adv Polvamine ,, Res 1983; 4: i21-31. 264. Bacchi CJ. Nathan HC. Hutner SH. McCann PP. Sioerdsma A. Novel 'combination chemotherapy of experimental trypanosomiasis by using bleomycin and DL-alphadifluoromethylornithine.Biochem Pharmacoll982;31: 28336. 265. Bacchi CJ, Garofalo J, Mockenhaupt D, McCann PP,

266.

267.

268. 269.

270. 271.

Diekema KA, Pegg AE, Nathan HC, Mullaney EA, Chunosoff L, Sjoerdsma A, Hutner SH. In vivo effects of alpha-DL-difluoromethylornithine on the metabolism and moroholoav of TrvDanosoma brucei brucei. Mol Biochem Parasitol 7983; 7: 209-25. Bitonti AJ. Bvers TL. Bush TL, Casara PJ, Bacchi CJ, Clarkson AB- Jr, McCann PP,. Sjoerdsma A. Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of Sadenosylmethionine decarboxylase. Antimicrob Agents Chemother 1990; 34: 1485-90. Giffin BF, McCann PP, Bitonti AJ, Bacchi CJ. Polyamine depletion following exposure to DL-alpha- difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondria1 activation in a monomorphic strain of Trypanosoma brucei brucei. J Protozool 1986; 33: 238-43. Sjoerdsma A, Schechter PJ. Chemotherapeutic implications of polyamine biosynthesis inhibition. Clin Pharmacol Ther 1984; 35: 287-300. Garafalo J, Bacchi CJ, McLaughlin SD, Mockenhaupt D, Trueba G, Hutner SH. Ornithine decarboxylase in Trypanosoma brucei: Evidence for selective toxicity of difluoromethylornithine. J Protozool 1982; 29: 389-94. Phillips MA, Coffino P, Wang CC. Cloning and sequencing of the ornithinedecarboxvlase aene from Trvpanosoma brucei. .. J Biol Chem 1987; 262;8721-7. Ghoda L. PhilliDs MA, Bass KE, Wang CC, Coffino P. Trypanosome orhithine 'decarboxylase isstable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation. J

259

Biol Chem 1990; 265: 11823-6. 272. Schechter PJ, Sjoerdsma A. Difluoromethylornithine in the treatment of African trypanosomiasis. Parasitol Today 1986; 2: 2 2 3 4 . 273. Whaun JM, Brown ND. Ornithine decarboxylase inhibition and the malaria-infected red cell: A model for polyamine metabolism and growth. J Pharmacol Exp Ther 1985; 233: 507-1 1. 274. Assaraf YG, Abu-Elheiga L, Spira DT, Desser H, Bachrach U. Effect of polyamine depletion on macromolecular synthesis of the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes. Biochem J 1987; 242, 221-6. 275. Assaraf YG, Golenser J, Spira DT, Messer G, Bachrach U. Cytostatic effect of DL-alpha-difluoromethylornithine against Plasmodium falciparum and its reversal by diamines and spermidine. Parasitol Res 1987; 73, 313-8. 276. Bitonti AJ, Dumont JA, Bush TL, Edwards ML, Stemerick DM, McCann PP, Sjoerdsma A. Bis(benzy1)polyarnine analogs inhibit the growth of chloroquine-resistant human malaria parasites (Plasmodium falciparum) in vitro and in conbination with a-difluoromethylornithinecure murine malaria. Proc Natl Acad Sci USA 1989; 86: 651-5. 277. Golden JA, Schoerdsma A, Santi DV. Pneumocystis carinii pheumonia treated with alpha-difluoromethylornithine. West J Med 1984; 141: 613-23. 278. Gilman TM, Paulson YJ, Boylen CT, Heseltine PNR, Sharma PO. Eflornithine treatment of Pneumocystis carinii pneumonia in AIDS. JAMA 1986; 256: 2197-8. 279. Fozard JR, Grove J, Part ML, Prakash NJ. Inhibition of early embryogenic deveiopment in mice by alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of Lornithine decarboxylase. Br J Pharmacol 1979; 66: 436P7P. 280. Fozard JR, Part ML, Prakash NJ, Grove J, Schechter PJ, Sjoerdsma A, Koch-Weser J. L-Ornithine decarboxylase: an essential role in early mammalian embryogenesis. Science 1980; 208: 505-8. 281. Fozard JR, Part M-L, Prakash NJ, Grove J. Inhibition of murine embryonic development of alpha-difluoromethylornithine, an irreversible inhibitor or ornithine decarboxylase. Eur J Pharmacol 1980; 65: 379-91. 282. Reddy PRK, Rukmini V. Alpha-difluoromethylornithine as a postcoitally effective antifertility agent in female rats. Contraception 1981; 24: 215-21. 283. Pasquali J-L, Mamont PS, Weryha A, Knapp A-M, Blervaque A, Siat M. Immunosuppressive effect of (2R, 5R)6-heptyne-2,5-diamine an inhibitor of polyamine biosynthesis: I: Effects on mitogen-induced immunoglobulin production in human cultured lymphocytes. Clin Exp lmmunol 1988; 72: 141-4. 284. Claverie N, Pasquali J-L, Mamont PS, Danzin C, WeilBousson M, Siat M. Immunosuppressive effects of 2 5 diamine, an inhibitor of polyamine synthesis. II. Beneficial effects on the development of a lupus-like disease in MRLl p r l l p r mice. Clin Exp lmmunol 1988; 72: 293-8. 285. Thomas TJ, Messner RP. Beneficial effects of a polyamine biosynthesis inhibitor on lupus in MRL-I prilpr mice. Clin Exp lmmunol 1989; 78: 239-44. 286. Wolos JA, Logan DE, BowlinTL. Methylacetylenicputrescine (MAP), an inhibitor of polyamine biosynthesis, prevents the development of collagen-induced arthritis. Cell lmmunoll990; 125: 498-507.

Ann Med 23

Cystitis: from urothelial cell biology to clinical applications.

Cocoa and Dark Chocolate Polyphenols: From Biology to Clinical Applications.

Ras in digestive oncology: from molecular biology to clinical implications.

Hyaluronidase: from clinical applications to molecular and cellular mechanisms.

Postischemic revascularization: from cellular and molecular mechanisms to clinical applications.

Leukocyte cell adhesion proteins: from molecular dissection to clinical applications.

The applications of synthetic oligonucleotides to molecular biology.

Applications of chaos theory to the molecular biology of aging.

signalling: biology and clinical applications.

Nutrition science from vitamins to molecular biology.

Molecular biology. recA: from locus to lattice.

Molecular biology and respiratory disease. 2-- Applications to the study and treatment of respiratory disease: methods in molecular biology.

Polyamines in mammalian biology and medicine.

Avian antimicrobial host defense peptides: from biology to therapeutic applications.

Research applications of proteolytic enzymes in molecular biology.

Molecular recognition with boronic acids-applications in chemical biology.

Applications of Discrete Molecular Dynamics in biology and medicine.

Physiology, molecular biology and applications of the bacterial starvation response.

Inflammatory therapeutic targets in coronary atherosclerosis-from molecular biology to clinical application.

Molecular targeted therapy in hepatocellular carcinoma: from biology to clinical practice and future.

Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell.

Diagnostic tools from molecular biology.

The CD34 antigen: structure, biology, and potential clinical applications.

Helicobacter pylori neutrophil-activating protein: from molecular pathogenesis to clinical applications.