Polyamines in Mammalian Tumors Part I

POLYAMINES IN MAMMALIAN TUMORS Part I Giuseppe Scalabrino and Maria E. Ferioli Institute of General Pathology and C.N.R. Centre for Research in Cell Pathology. University of Milan. Milan, Italy

Nil minus est hominis occupati quam vivere: nullius rei difficilior scientia est. Professores aliarum artium vulgo multique sunt, quasdam vero ex his pueri admodum ita percepisse visi sunt, ut etiam praecipere possent: vivere tota vita discendum est et, quod magis fortasse miraberis, tota vita discendum est mori. SENECA, “De Brevitate Vitae,” 7, 3 L’ignorance qui estoit naturellement en nous, nous l’avons, par longue estude, confirmbe e t averbe. MONTAIGNE, “Essais,” L. 11, C. 12

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I. Introduction and Background , , , . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structure-Function Relationship of Polyamines , . . . . . . . . . . . . . . .. .. B. Properties of the Biosynthetic Enzymes of Polyamines . . . . . . . . . . . . . C. S-Adenosyl-L-Methionine . . . . . . . . . . . . . . . , , . .. . . . . .. .. .. . D. 5’-Methylthioadenosine , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Catabolism of the Major Polyamines in Mammalian Organisms . . . F. Conjugation Products and Excretion Products . . . . . . . . . . . . . . . . . . . . . . . G. Natural Antipolyamine Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . 11. Levels of Polyamines and Their Biosynthetic Enzymes in Fully Developed Experimental Tumors . . . .. . . . . . . . , . . . . . . . . . . . , . . . . . . . . . . . . . . . 111. Modification in Vivo and in Vitro of Tissue Polyamine Metabolism by Chemical Carcinogens and Tumor Promoters . . . . . . . . . . . . . . . . . . . . . . . A. Effects of a Single Administration of a Carcinogen or Tumor Promoter on Polyamine Biosynthesis and Content in the Target Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects of Repeated or Prolonged Administration of a Carcinogen or Tumor Promoter on Polyamine Biosynthesis and Content in the Target Tissues.. . . . . . . . . . . . . . . . . . . . . . . .. . . . C. Multistage Carcinogenesis , . . . . . . , . . . . . . . . . . . . . , , . . . . . . . . . . . ..... D. Mutagenic Action and Antimutagenic Properties of Polyamines . . . . . . . . E. Polyamine Levels in Urine, Sera, and Erythrocytes of Rats during Chemical Carcinogenesis or Bearing Several Experimental Tumors . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Biosynthesis and Levels of Polyamines in Cells during the Virus-Induced Transformation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Effects of Oncogenic RNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects of Onbgenic DNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Changes in Polyamine Biosynthesis and Content of Target Tissues by Physical Carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

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Copyright @ 1981 by Academic Press,Inc. All rights of reproduction in any form resewed. ISBN 0-12-006635-1

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I. Introduction and Background

Afker the discovery of polyamines, it became clear in time that they are ubiquitous in mammalian body tissues. Moreover, an everincreasing number of important regulatory functions have been attributed to the polyamines in the different fields of cellular biochemistry in eukaryotes. Excellent reviews of the physiological functions fulfilled by polyamines, or of some particular aspects of these, are available in the enormous literature on these substances (Tabor et al., 1961; Tabor and Tabor, 1964, 1972; Bachrach, 1970b; Raina and Janne, 1970a, 1975; Stevens, 1970; Smith, 1971, 1972; Cohen, 1972; Williams-Ashman, 1972; Williams-Ashman et al., 1972a, 1973; Morris and Fillingame, 1974; Barbiroli et al., 1976; Caldareraet al., 1976; Cli, et al., 1976; Russell et al., 1976a; Sakai and Cohen, 1976c; C. W. Tabor and Tabor, 1976; Karpetsky et al., 1977; Pardee et al., 1978; Canellakis et al., 1979; Cohen and McCormick, 1979; Maudsley, 1979; Quash and Roch, 1979; Stevens and Winther, 1979; Williams-Ashman and Canellakis, 1979; Heby and Andersson, 1980; McCann, 1980; Russell, 1980; Theoharides, 1980). Furthermore, the roles of polyamines in the biochemistry of particular organs or metabolic pathways are included in some monographs or reviews (Raina, 1963; Janne, 1967; Siimes, 1967; Williams-Ashman et al., 1969, 1976, 1980; Janne et al., 1976; Algranati and Goldenberg, 1977; Kramer et al., 1979; Shaw, 1979; Slotkin, 1979; Lesiewicz and Goldsmith, 1980; Lowe, 1980). Three comprehensive books have also been published, by Cohen ( 1971), Bachrach (1973), and Gaugas (1980a). A series of volumes, of which the two that have appeared so far were edited by Campbell et al. ( 1978a,b), present recent advances in the polyamine field in a variety of disciplines, and thus indicate the growing importance of polyamines in biochemistry and medicine. The proceedings of two symposia have been edited by Kremzner (1970) and Herbst and Bachrach (1970), in which most of the information about the metabolism and function of polyamines obtained before 1970 is elucidated. Finally, there is one short, but critical, article (Cohen, 1978) about particular biochemical functions of polyamines. Although many of the reviews cited include one (generally short) paragraph on the polyamines in mammalian tumors, only a symposium volume edited by Russell (1973a) and one book more recently published by Russell and Durie (1978), and five reviews by Russell (1973b,c), Bachrach (1976b), Janne et al. (1978), and Milano et al. (1980) are devoted to the data accumulated during the past ten years on this topic and to highlighting the behavior and significance of

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polyamines and their biosynthetic enzymes in oncology, in both animals and humans. The present review is intended not as a comprehensive summary nor, even less, as an analytical discussion of the different aspects of the biochemistry and physiology of polyamines in the different types of living organisms. For an in-depth discussion of those subjects we refer the reader to the above-mentioned references. However, in this introductory section some material concerning the structure, distribution, and function of polyamines, almost exclusively limited to mammalian cells, will b e included. General information about the biosynthetic and degradative pathways of the main polyamines in mammalian cells will also be up-dated. This we consider to be a necessary and useful preamble to the sections that follow, which will be strictly devoted to mammalian neoplasms and in which some concepts about the physiology of polyamines and the regulation of their metabolism in normal cells will be discussed. Our aim is to review selectively and in detail the extensive literature on the connections between the polyamines and cancer in mammals and to judge whether these connections are coincidental or characteristic of the neoplastic disease process. Because of the numerous recent developments in this area, another purpose of the present review is to appraise its present status. A customary and widely accepted definition of a tumor is that it is a mass of abnormal cells with altered phenotypes; its many differences from normal cells, in both morphological and biochemical features, lead to excessive growth of the tumorous cells. Thus, the essence of the concept of neoplasm is the seemingly autonomous and uncontrolled cell multiplication. Consequently, the modifications in polyamine biosynthesis and content observed in several biological models of controlled cell growth in mammals-such as liver regeneration, regenerating nerves, different target organs after treatment with growthpromoting hormones, myocardial hypertrophy, kidney hypertrophy, prostatic hyperplasia, wound healing, EGF-stimulated cultured epidermal cells or fibroblasts, mitogen-stimulated cultured lymphocytes, and NGF-stimulated neurons-will be deliberately left out of this review; the reader will find the appropriate references in the reviews and books cited.

A. STRUCTURE-FUNCTION RELATIONSHIP OF

POLYAMINES

The aliphatic polyamines are nonprotein, polycationic substances that are widely distributed in living organisms (animals, bacteria, vi-

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ruses, yeasts, and plants) and in biological fluids. The main physiological polyamines present in different types of tissues are putrescine and its propylamine derivatives, spermidine, and spermine. Cadaverine and 1,Sdiaminopropane are found only rarely in animal tissues and in biological fluids. The common name of putrescine is derived from its first isolation from cultures of Vibrio cholerae and from cadavers undergoing bacterial decomposition, and that of spermine to the discovery of spermatozoa together with crystals of the phosphate salt of spermine in human seminal fluids (Cohen, 1971).During synthesis of spermine, another base, which was identified as spermidine phosphate, was obtained. From a chemical point of view, the term “polyamines” is, in one respect, a misnomer, since it suggests a compound with multiple amine residues. To circumvent this problem, it has been suggested recently that the term polyamines can be replaced with the term “oligoamines,” which is chemically more precise (Stevens and Winther, 1979). Putrescine is, strictly speaking, a simple diamine, whereas spermidine is a triamine and spermine a tetraamine. However, the metabolic and functional similarities of these compounds are so close that it is convenient and conventional to classify and handle them together. The formulae of the aforementioned polyamines are as follows: HzNCHZCH2CHJVHZ 1,3-Diaminopropane

H2NCH2CHzCHZCHZNHz 1,4-Diaminobutane or 1,CTetramethylenediamine (putrescine) HzNCHpCHzCHzCH&HZNH2 l,5-Diaminopentane (cadaverine)

HZNCHZCHZCH~NHCH&H&H&H~NH~ N-(Spropylamine)-1,4-diaminobutane or 4-Azaoctane-1,8-diamine or N-(3-aminopropy1)-tetramethylene-1,Cdiamine (spermidine)

HJVCHzCHzCHzNHCHzCHzCHzCHzNHCH&HzCHzNHz

N,N‘-bis(3-propylamine)-1,4-diaminobutaneor 4,9-Diazadodecane-l,12-diamine or N,N’-bis(3-aminopropyl)-tetramethylene-l,4-diamine (spermine)

The polyamine pattern may vary markedly from one species to another, and in a given species it differs greatly among various tissues and also depends on growth conditions, growth rate, age, etc., of the tissue. In general, spermidine and spermine are the main polyamines synthesized in eukaryotes, usually present in millimolar concentrations, whereas smaller amounts of putrescine are synthesized, usually in nanomolar concentrations. Prokaryotes have higher concefitrations of putrescine than spermidine and lack spermine, except for a few

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bacterial species. Thus, spermine synthesis appears to be either very rare or utterly lacking in bacteria, suggesting that spermine has some nuclear function. Using mouse cells, it was found that, although spermidine and spermine occur in both nuclear and cytoplasmic fractions, the concentration of these t w o polyamines in nuclei surrounded by membranes (the so-called karyoplasts) is at least three times higher than that in enucleated cells (the so-called cytoplasts) or in whole cells (Clark and Greenspan, 1977; McCormick, 1977, 1978a). The ability of nucleated eukaryotic cells to synthesize spermine may be considered to be an evolutionary acquisition of function in which spermidine is converted to a novel functional compound by the addition of an aminopropyl moiety. However, an intriguing, but still unsolved, question is whether spermine has its own qualitatively specific properties in the cell biochemistry of eukaryotes, such that it cannot be replaced at all by spermidine, or whether it is a duplicate of spermidine and consequently could be considered to be a bonus biomolecule. Unfortunately, although several mutants lacking in one of the polyamines or various polyamine biosynthetic enzymes have been isolated and characterized from yeasts and bacteria (Hirshfield et al., 1970; Maas et al., 1970; Morris and Jorstad, 1970, 1973; Young and Srinivasan, 1972, 1974; Morris and Hansen, 1973; Algranati et al., 1975; CunninghamRundles and Maas, 1975; Echandi and Algranati, 1975a,b; Goldemberg and Algranati, 1977; Hafner et al., 1977,1978,1979; Cohn et al., 1978a,b,c, 1980; Geiger and Morris, 1978a,b; Taboret al., 1978; Whitney and Morris, 1978; Whitney et al., 1978; Igarashi et al., 1979), no analogous mutants of any mammalian cell lines are available. Only mutant mammalian cell lines showing decreased polyamine transport have been described (Mandel and Flintoff, 1978). In eukaryotes, however, few instances have been found thus far in which spermidine and spermine are not interchangeable to some degree. Throughout the growth of fish and rat brain and in the livers of mice starved or treated with phenobarbital, spermine levels correlate with those of DNA, whereas spermidine levels show excellent correlation with the RNA content in the tissue (Seiler et al., 1969; Seiler, 1973; Seiler and Lamberty, 1975; Seiler and Schmidt-Glenewinkel, 1975). Analogous localizations of spermidine and spermine have been found in viruses. In fact, in herpes simplex virus type 1 (HSV-l), which mature their virions in the nucleus of the infected cell, spermine is associated with the nucleocapsid, whereas spermidine is isolated with the viral envelope (Gibson and Roizman, 1971; Cohen and McCormick, 1979); in vaccinia virus, which, like pox viruses, multiplies exclusively in the cytoplasm of the infected cell, spermine is associated with the DNA-

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containing cores and spermidine with the membrane material (Lanzer and Holowczak, 1975). Furthermore, in explants of mouse mammary gland, spermidine but not spermine can replace glucocorticoids in stimulating the synthesis of a-lactalbumin and casein (Oka, 1974; Oka and Perry, 1974a,b; Kano and Oka, 1976; Rillema et al., 1977; Houdebine et al., 1978; Oka et al., 1978a; Bolander and Topper, 1979). However, it has been recently demonstrated that this action of spermidine in mammary gland seems to differ from species to species (Bolander and Topper, 1979). Finally, it has been reported that the rat ventral prostate contains an acid protein that is localized in the cytosol, is induced by androgens, and binds spermine much more tightly than spermidine or other natural diamines (Liang et al., 1978; Liao et al., 1979; Mezzetti et al., 1979). Just recently, in order to differentiate between those effects of spermine due to its polycationic nature and its effects as a specific component needed for maximum efficiency in protein synthesis, an immunochemical approach has been developed (Quash et al., 1976; Bartos and Bartos, 1978; F. Bartos et al., 1979, 1980; Niveleau and Quash, 1979). Using specific antispermine antibodies, an inhibition of protein synthesis in the wheat germ cell-free system programmed with exogenous mRNA was shown and was found to be specific, since it was not overcome by putrescine or spermidine but only by spermine (Niveleau and Quash, 1979). In addition to the aforementioned polyamines, another group of polyamines has been recently identified in plants, algae, bacteria (especially thermophilic bacteria), some multicellular marine organisms (e.g., arthropods), and mammals (Johnson and Markhan, 1962; Kullnig et al., 1970; Kuttan et al., 1971; Imaoka and Matsuoka, 1974; Oshima, 1975, 1979; De Rosa et al., 1976, 1978, 1980; Nickerson and Lane, 1977; Stillway and Walle, 1977; Kneifel et al., 1978; Rosano et al., 1978; Zappia et al., 197813; Aleksijevic et al., 1979; Kremzner and Sturman, 1979; Yamamoto et al., 1979). Among these so-called new polyamines it seems important to mention the following compounds: caldine, thermine, and thermospermine. The formulae of these new polyamines and of other polyamines of minor importance are as follows: HzNCHzCHOHCHpCHSNHi 2-Hydroxyputrescine

HpNCHzCHOHCHzCHzNHCHzCHzCHzNHz

2-H ydroxy spermidine HSNCH~CH&H&JHCH&~HZCH~NH,

Sym-norspermidine or Bis(3-aminopropy1)amine or 1,7-Diamino-4-azaheptane (caldine)

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HzNCH&HzCHzCHzNHCHZCHzCH2CHzNHz Bis(4-aminobuty1)amine(homospermidine)

HOOCCHzCHzNHCH2CHzCH&HzNHCHzCHzCOOH

N , N '-bis(2-carboxyethyl)-1,4-diaminobutane(spermic acid)

H~NCH&HZCH~NHCH~CHZCH~NHCH&HZCH~NH~ 1,l l-Diamino-4,8-diazaundecaneor N,N'-bis(3-aminopropyl)-1,3-diamino-propane or Norspermine or Sym-norspermine or N,N'-bis(3-aminopropyl)-tri-methylene-1,3-diamine (thermine)

H~NCH~CH~CH~NHCH~CH~CH~NHCH~CH~CH~CH~NH~ 1,12-Diamino-4,8-diazododecane(thermospermine)

Although some of these new polyamines have also been found in eukaryotes, their biological significance and specificity in these organisms remains to be clarified. In mammals, although remarkable amounts of polyamines are present in the main biological fluids (especially in seminal fluid), these substances fulfill their paramount biochemical roles inside the cell. Most of the natural functions of polyamines in different kinds of biological systems are closely connected with their physicochemical properties. The three most widely distributed polyamines (putrescine, spermidine, and spermine) are very soluble in water, have weak chelating capacity, and are stable polycations with pK values between 8.0 and 11.0 for the primary amino group and between 9.0 and 10.9 for the secondary amino group. At physiological pH, i.e., around neutrality, all of the primary and secondary amino groups of putrescine, spermidine, and spermine are protonated, so that they have two, three, or four basic centers, respectively. Most of the various biological effects of these polyamines are undoubtedly related to this polybasic structure. In fact, polyamines are known to have high affinity for negatively charged compounds and molecules, e.g., the phospholipids of cell membranes and of myelin-rich structures and nucleic acids (Ames and Dubin, 1960; Kropinski et al., 1973; Levy et al., 1974; Gabbay et al., 1976; Gosule and Schellman, 1976, 1978; Osland and Kleppe, 1977; Bolton and Kearns, 1978; Chattoraj et al., 1978; Damaschun et al., 1978; Giorgi, 1978; Minyat et al., 1978; Becker et al., 1979). In uitro, polyamines directly stimulate various DNA and RNA polymerases, methylases, nucleotidyltransferases, hydrolases, and ribonucleases and affect reactions involving tRNA, ribosomal RNA, and mRNA molecules, thus contributing to polyribosomal protein synthesis (Janne et al., 1976; Canellakis et al., 1979; Cohen and McCormick, 1979; Williams-Ashman and Canellakis, 1979). Therefore, polyamines appear to be implicated in virtually every step of the synthesis and ultimate metabolic fate of RNA. In the sequence DNA+ RNA+ protein the polyamines influence, at the transcriptional level, strand

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selection and chain initiation, extension, or termination and, at the translational level, preparation of tRNA, aminoacylation, messenger binding to the ribosomal subunits, the translation of natural and synthetic mRNAs, and the assembly of the ribosomal subunits (Raina and Janne, 1970a; Barbiroli et al., 1976; Caldarera et al., 1976; Janne et al., 1976; Maudsley, 1979). Additionally, polyamines stabilize the structures of tRNA, ribosomes, and polysomes and promote the attachment of free ribosomes to endoplasmic reticular membranes (Cohen, 1971; Bachrach, 1973; Janne et al., 1976; Cohen and McCormick, 1979; Maudsley, 1979).Always because of the polybasic characteristic of the polyamines, these nitrogenous bases are known to specifically bind to the polyanionic nucleate, thus stabilizing nucleic acids against denaturation and digestion by nucleases (Cohen, 1971; Bachrach, 1973; Janne et al., 1976; Williams-Ashman and Canellakis, 1979). The multitudinous effects that spermidine and spermine have in cell-free systems that synthesize or degrade nucleic acids or proteins may be both stimulatory and inhibitory, depending not only on the concentrations of the polyamines but also on the experimental conditions, such as ionic strength (particularly of Mg ions), pH, and temperature (Raina and Janne, 1975; C. W. Tabor and Tabor, 1976). Although no direct evidence proves that polyamines have these same functions in vivo in intact cells, a large body of indirect evidence suggests that the close association between nucleic acids and polyamines also exists in vivo in prokaryotic and eukaryotic organisms. Another important feature of the molecular structure of polyamines is the possibility of rotation around the carbon-carbon and carbonnitrogen bonds, which confers considerable conformational flexibility to the polyamines (Sakai and Cohen, 1976c; Cohen, 1978; Cohen and McCormick, 1979). Therefore, spermidine and spermine can assume an extended conformation (as in the case of condensation with DNA) or a more locked conformation (as in the case of binding to tRNA) (Sakai and Cohen, 1976c; Cohen, 1978; Cohen and McCormick, 1979).X-Ray analysis has suggested several possible configurations for the complex formed between the polyamines and double-stranded DNA. Spermidine and spermine can bind to DNA through the interactions of phosphate groups with each positively charged amino group, the tetramethylene portion of the polyamine bridges the narrow (minor) groove of the helix between the two strands, and the trimethylene portion bridges adjacent phosphate groups (Sakai and Cohen, 1976c; Cohen, 1978; Cohen and McCormick, 1979). Alternatively, polyamines can bind in the minor groove of' DNA by hydrogen bonding of the amines to phosphate oxygens (Sakai and Cohen, 1976c;

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Cohen, 1978; Cohen and McCormick, 1979). It is likely that all these configurations can be adopted in viuo in either a random or nonrandom manner, so that the polyamines can be attached like the histones along sections of DNA molecule. According to what has been demonstrated so far, the role of spermine and spermidine in gene expression seems to lie in the induction of supramolecular structures of the DNA, in competition with the histones and nonhistone proteins, or of the RNAs in RNA-protein complexes. The secondary and tertiary structures of all sorts of polynucleotides can be profoundly affected by polyamines. Molecules of tRNA can be converted from their inactive configuration to a more compact active form by the addition of monovalent cations or of much lower concentrations of Mg2+or polyamine. Moreover, specific tight binding sites for spermidine exist in the tRNA molecules of yeast and Escherichia coli (Ladner et al., 1972; Pochon and Cohen, 1972; Santi and Webster, 1975; Takeda and Ohnishi, 1975; Evans and Deutscher, 1976; Prinz et al., 1976; Bolton and Kearns, 1977a,b; de Varebeke and Augustyniak, 1977). It seems opportune to mention here that recent crystallographic studies have defined the position of spermine within the molecule of yeast phenylalanine tRNA (Cohen, 1978; Quigley et al., 1978). This is probably the first example of elucidation of the function of a polyamine in terms of molecular structure. However, differences in experimental results, probably due to the different experimental conditions, have been obtained, and their interpretations are still the object of discussion. In many aspects, the polyamines may be regarded functionally as a group of compounds acting like organic cations (Veloso et al., 1973; Igarashi et al., 1975; Imai et al., 1975; Fukuyama and Yamashita, 1976; Nakai and Glinsmann, 1977b; Tanigawa et al., 1977; Lovgren et al., 1978; Kitada et al., 1979). In many in vitro systems studied so far, the effectiveness of the polyamines follows a cationic progression, i.e., spermine, the strongest base, is the most effective, followed by spermidine and then by putrescine, whereas in a few other studies a precise structural demand for a specific polyamine has been shown. Interchangeability between polyamines and Mg2+ and other cations has been described in several steps of protein biosynthesis, albeit Mg2+may not be able to substitute for all the polyamine molecules (Khawaja and Raina, 1970; Khawaja, 1971, 1972). It has been shown that M$+ ions can substitute, to some extent, for spermidine and spermine in the process of DNA synthesis in cultured cells (Melvin and Keir, 1979). In the case of yeast tRNAPhe,previously mentioned, the role of polyamine, which is to modify the structure of the mole-

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cule by pulling strands together and producing bends in particular regions, is quite different from that of Mg2+ (Cohen, 1978; Cohen and McCormick, 1979). Similarly, the binding of M$+ to a nucleotide is strictly one to one, whereas interactions between polyamines and nucleotides are affected not only by charge interactions but also by structural features of polyamine molecules, resulting in variable and multiple binding. The polyamines can also compete with M g + for complex formation with AMP, ADP, ATP, and other nucleotides. There are other biochemical reactions in which Mg2+ and polyamines are not interchangeable. Mg2+ and spermine within certain concentration ranges seem to be synergistic in elevating the respiratory control ratio associated with mitochondria1 oxidation of P-hydroxybutyrate, but the spermine concentrations involved are much lower than those of Mgz+ (Chaffee et al., 1977, 1978, 1979). Moreover, spermine cannot completely substitute for MgZfin enhancing the phosphorylation of nonhistone chromatin proteins (Imai et al., 1975) or in stimulating the choline kinase reaction (Fukuyama and Yamashita, 1976), and the inhibiting effect of spermine on lipid peroxidation is much stronger than that of Mgz+(Kitada et al., 1979). Finally, spermine promotes the ADP-ribosylation of nonhistone proteins, whereas Mg2+promotes that of histones (Tanigawa et al., 1977). Functionally, the polyamines offer a selective advantage over inorganic cations in that intracellular synthesis is possible. This permits fine adjustments of the intracellular polyamine concentrations according to different physiological conditions. However, a set of very complex systems for maintaining the cellular homeostasis of magnesium and other metal cations does exist, although many aspects of it are yet undefined (Rasmussen and Bordier, 1974). In the case of magnesium, this is demonstrated by the facts that mammalian cells are able to retain nearly normal MgZ+content despite extracellular Mg2+deficiency and that, conversely, Mg2+uptake declines when hypermagnesemia develops (Rasmussen and Bordier, 1974). Unlike polyamines, the MgZ+content of the mitochondria is generally greater than that in the cytosol (Rasmussen and Bordier, 1974). Within cells, MgZfand other metal cations are obtained from the bloodstream, whereas polyamines are produced within the cells and then released into the blood stream. However, there is an important point of contact between the cellular regulation of polyamine biosynthesis and Mg2+concentration in that both are under the influence of several hormones. Naturally occurring aliphatic polyamines have been shown to influence a number of biochemical reactions involving membrane functions. Several authors, using membranes purified from different tissues,

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have reported that spermine and spermidine inhibit Na+,K+-ATPase activity, which is generally considered to be the enzymic mechanism of cation transport across the cell membrane (Ahmed and WilliamsAshman, 1969; Peter et al., 1973; Tashima and Hasegawa, 1975; Heinrich-Hirsch et al., 1977; Quarfoth and Ahmed, 1977; Tashima et aZ., 1977, 1978; Quarfoth et aZ., 1978). A similar inhibitory effect of spermine and spermidine on the activity of CaZ+,Mg2+-ATPasehas been shown in the muscular tissue (De Meis and De Paula, 1967; Nagai et al., 1969), in which polyamines also promote the polymerization of actin (Oriol-Audit, 1978). Therefore, it is quite possible that the polyamines are involved, among others, in physiological contraction processes, e.g., as regulators of ATP concentrations. Interestingly enough, a regulatory effect on membrane-bound acetylcholinesterase has also been demonstrated (Kossorotow et al., 1974; Anand et al., 1976; Max and Oh, 1977), hinting that the polyamines may also have a role in synaptic transmission. Polyamines have been found to have important effects on different membranes with which they are possibly associated in uiuo. A marked stabilization of membrane structure against lysis or swelling has been reported for several microorganisms and mammalian subcellular fractions (Mager, 1959; Anderson and Norris, 1960; Herbst and Witherspoon, 1960; Tabor, 1960; Quigley and Cohen, 1969). The mechanism of this stabilization remains to be elucidated, but it is conceivable that the polyamines, as polycations, strongly bind to membranes, which usually have a net negative charge. A further molecular explication for such stabilization may reside in the recently reported demonstration that polyamines markedly protect mitochondria1 membranes against the destabilizing action of exogenous phospholipases (Sechi et al., 1978). It is well known, in fact, that the endogenous phospholipases bound to many membranes, such as those of lysosomes and mitochondria, operate in vivo to seriously alter membrane structure. During recent years, new biochemical reactions in the regulation of which the polyamines are physiologically involved have been identified. Thus, we attach paramount importance to the fact that these substances can act as modulators of the metabolism of cyclic nucleotides, of the activities of protein kinases, and of glycerolipid biosynthesis. The role of polyamines in the biosynthesis and the inactivation of the cyclic nucleotides will be discussed in detail in Part 11, Section I of this article in Volume 36. The effect of polyamines on the activity of protein kinases in normal cells depends on the type of protein kinase tested (Imai et aZ., 1975; Lee and Iverson, 1976; Murray et al., 1976; Takai et aZ., 1976; Maen-

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paa, 1977; Nakai and Glinsmann, 1977a; Ahmed et al., 1978; FarronFurstenthal and Lightholder, 1978; Hochman et aZ., 1978; Job et d., 1979). Polyamines have been shown to inhibit the activity of CAMPdependent protein kinases and, on the contrary, to stimulate the activities of both cGMP-dependent and cyclic nucleotide-independent protein kinases (Lee and Iverson, 1976; Murray et al., 1976; Takai et al., 1976; Maenpaa, 1977; Hochman et al., 1978; Job et al., 1979). When such regulation involves the phosphoprotein kinases localized in the nucleus, this would mean that the polyamines could also assume a regulatory role in genetic expression, since it is well established that nuclear kinases have a key role in the regulation of chromosomal protein phosphorylation and gene expression. Germane to the stimulation by spermine and spermidine of the chromatin-associated noncyclic AMP-dependent protein kinases are the reports that polyamines can directly enhance certain nucleoside diphosphate kinase reactions (Nakai and Glinsmann, 1977a) and the aforementioned choline kinase reaction (Fukuyama and Yamashita, 1976). Finally, studies have recently reported that spermidine and spermine stimulate the synthesis of triglyceride (Jamdar, 1977, 1978).

B. PROPERTIES OF THE BIOSYNTHETIC ENZYMES OF POLYAMINES Four enzymes are known to be involved in the biosynthesis of polyamines in eukaryotic organisms: two decarboxylases and two synthases. L-Ornithine decarboxylase (L-ornithine carboxyl-lyase, EC 4.1.1.17) catalyzes the formation of putrescine from L-ornithine. S-Adenosyl-L-methionine decarboxylase (SAMD) (EC 4.1.1.50) produces S-methyladenosylhomocysteamine (“decarboxylated Sadenosylmethionine,” also defined as S-adenosyl-(5’)-3-methylthiopropylamine) from S-adenosyl-L-methionine (SAM). This enzyme is of paramount importance for the syntheses of spermidine and spermine. Spermidine synthase catalyzes the transfer of the propylamine group to putrescine to yield spermidine. Spermine synthase transfers the propylamine group from S -methyladenosylhomocysteamine to spermidine to form spermine. In both the latter reactions, methylthioadenosine and one proton are the other reaction products. For these four enzymes, a large body of experimental evidence points out the key role of enzyme ornithine decarboxylase (ODC) as the rate-limiting step in the biosynthetic pathway of the polyamines. The scheme for the biosynthesis of putrescine, spermidine, and spermine in mammalian tissues is presented in Fig. 1. The closely related connections between the biosynthetic pathway of polyamines

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OR

S - METHYLADENOSYL-HOMOCYSTEAMlflE OR

S -ADENOSYL - (5') - 3 PHZ N&C,C/N\ I I HC+N/c\NGH

SPERMIOINE CH (CHoH),C

L - 0 1 5'- MET HY LT H 10ADENOSINE + H+

SPERM INE

@ L-ORNITHINE CARBOXY-LYASE (ORNITHINE DECARBOXYLASE) (EC 4 1 1 17) @ ATP L-METHIDNINE -S-ADENOSYLTRANSFERASE(S-ADENOSYLMETHIONINE SYNTHETASE)(EC2 5 16)

0

@

@

S-AOENOSYL-L-METHIONINE CARBOXY-LYASE (S-ADENOSYL-L-METHIONINE DECARBOXYLASE)(EC41150) S-METHYLADENOSYLHOMOYSTEAMINE PUTRESCINE AMINOPROPYLTRANSFERASE (SPERMIDINE SYNTHASE) (EC 2 5 1 16) S-METHYLAOENOSYLHOMOCYSTEAMINE SPERMlOlNE AMINOPROPYLTRANSFERASE (SPERMINE SYNTHASE )

FIG.1. Biosynthetic pathway of the chief polyamines in mammalian tissues.

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and some other major pathways of intermediary metabolism in mammalian cells are shown in Fig. 2. A second distinct pathway of putrescine biosynthesis is present in bacteria; in this pathway, agmatine is formed from arginine by arginine decarboxylase and, in turn, is hydrolyzed to putrescine and

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PROLINE

A’-PVRROLINE - 5CARBOXVL AT E

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CARBAMVL - PHOSPHATE GLUTAMIC GLUTAMIC A C I D +~-SEMIALDEHVDE

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7L-oRNITHINE

CYSTATHIONINE +CVSTEINE

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DECAREOXVLATED ADENOSVLMETHIONINE

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FUMARAT E

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urea by agmatine ureohydrolase (Bachrach, 1973; C. W. Tabor and Tabor, 1976). 1. Ornithine Decarboxylase Mammalian ODC is a pyridoxal phosphate-requiring enzyme, like most of the decarboxylases, and shows a specific requirement for thiols, with certain dithiols, such as dithiothreitol, being especially effective. In the absence of thiol compounds, the enzyme appears to polymerize and loses enzymic activity. The enzyme is specific for L-omithine, with an apparent K, value for its substrate between 0.1 and 0.2 mM. D-Omithine is not attacked. Mammalian ODC from several rat tissues has been extensively purified, and its principal molecular properties have been characterized. The basal ODC activity of most resting adult mammalian tissues is extremely low, with the notable exception of the prostate. Most ODC activity is localized in the soluble cytosol, only a little being detectable in some cellular organelles and in the nucleus (Pegg and Williams-Ashman, 1968a; Ono et al., 1972; Richman et al., 1975; Eloranta et al., 1976a; Murphy and Brosnan, 1976; McCormick, 1977, 1978a; Ferioli et al., 1980). ODC displays a well-documented circadian rhythm in many organs of the rat so far investigated, thus providing a good model for studies of enzymic chronobiology (Hayashi et al., 1972; Stone et al., 1974; Walker and Potter, 1974; Nicholson et al., 1976; Fujimoto et al., 1978; Noguchi et al., 1979; Scalabrino et al., 1979a; Yarygin et al., 1979). In the rat, the fine regulation of the circadian rhythm seems to reside, at least in part, in the pineal gland (Scalabrino et al., 1979a; Yarygin et al., 1979). Moreover, the circadian rhythm and the activity of ODC can be influenced by external cyclical habits and environmental conditions, such as the alternation of food-starvation and/or light-dark periods, and by diet composition (Hopkins et al., 1973; McAnulty and Williams, 1975, 1977; Yanagi et al., 1975; Maudsley et al., 1976; Farwell et al., 1977; Morrison and Goldsmith, 1978; Rozovski et al., 1978). Two remarkable attributes of mammalian ODC must be mentioned here. 1. In most adult organs so far investigated, the ODC activity can be modulated simultaneously in vivo by several hormones, which are organ specific. When a hormone, whatever its chemical nature, has an anabolic and/or differentiating effect on its target organ, the increase in ODC activity is an early event in the cellular response to the hormone. Very frequently, similar enhancement of the ODC activity also occurs in several mammalian tissues in response to other nonhormonal agents

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that can stimulate cell growth and/or induce cell differentiation. All these experimental facts imply a striking capacity of ODC to be induced. Such an increase in the levels of ODC activity could also be part of the coordinated growth response of a cell, i.e., of the so-called pleiotypic program (Hershko et al., 1971). The inducibility of ODC activity by hormonal and nonhormonal effectors has also been confirmed in isolated organs, as well as in cultured or isolated cells (Cohen et al., 1970; Aisbitt and Barry, 1973; Mallette and Exton, 1973; Hogan et al., 1974a; Antony et al., 1976; Haselbacher and Humbel, 1976; Lumeng, 1976, 1979; Nissley et al., 1976; Oka and Perry, 1976; Friedman et al., 1977b; Jefferson and Pegg, 1977; Jefferson et al., 1977; Osterman and Hammond, 1977, 1978, 1979; Scheinman and Burrow, 1977; Scheinman et al., 1977; Spaulding, 1977; D’Amore et al., 1978; Levine et al., 1978; Ostermanet at., 1978; Rupniak and Paul, 1978; Sakaiet al., 1978; Smith and Stange, 1978; Yanget al., 1978; Piik et al., 1979; Takigawa et al., 1979, 1980; Veldhuis and Hammond, 1979; Veldhuis et al., 1979, 1980; Kapyaho, 1980; Klingensmith et al., 1980; Lin et al., 1980; Parker and Vernadakis, 1980). Interestingly enough, it has been demonstrated using enucleated cells that ODC activity can be induced in the cytoplasts but not in karyoplasts and, more important, that the kinetics of ODC induction in the cytoplasts are similar in time course to those in the whole cell (Clark and Greenspan, 1979). On the contrary, when a hormone is antianabolic, a rapid decrease in ODC activity in the target tissue has been reported, as noticeably occurs in the lymphatic part of rat thymus when glucocorticoids are given (Richards, 1978; Scalabrino et al., 197913). In the majority of biological systems so far tested, the induction of ODC activity appears to be the result of the synthesis of new enzymic proteins and not of the activation of preexisting enzyme molecules, since it is prevented or at least greatly reduced by concomitant use of some inhibitors of protein and/or RNA syntheses and since it is simultaneous with an increase in the amount of the immunoreactive enzyme protein (Holtta, 1975; Canellakis and Theoharides, 1976; Scheinman et ul., 1977). However, several studies carried out in experimental systems under growing and nongrowing conditions, both in vivo and in vitro, have suggested the existence of another path of ODC induction, in which no new mRNA synthesis is required. The induction of ODC activity in these instances is completely or partially actinomycin D resistant; the ODC activity can even be stimulated by the drug (Russell and Snyder, 1969; Fausto, 1971; Kay and Cooke, 1971; Beck et al., 1972; Kay et al., 1972; Kay and Lindsay, 1973b; Clark, 1974; Hogan et al., 1974a,b; Byus and Russell, 1975; Oka and Perry, 1976;

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Yamasaki and Ichihara, 1976; Chen and Canellakis, 1977; Costa, 1978b; Grillo et al., 1978b; Lau and Slotkin, 1979; Inderlied et al., 1980; Lin et al., 1980; Perry and Oka, 1980). Therefore, ODC biosynthesis seems to be able to happen either contingently or not contingently on prior production of new ODC mRNA molecules and to be separately regulated at both transcriptional and translational levels (Heby and Emanuelsson, 1979). The underlying mechanisms by which a wide variety of factors stimulate mammalian ODC activity are still unclear. Some experiments with biological systems under growing conditions both in vivo and in vitro have been claimed to show that increased intracellular levels of CAMP are necessarily involved in this process. In turn, cAMP activates intracellular protein kinase. In more detail, ODC induction can occur if protein kinase type I alone is stimulated; a subsequent activation of protein kinase type I1 does not seem to be necessary. After the activation of protein kinase, the synthesis of new mRNAs follows, and, on translation of the newly synthesized ODC mRNAs, there is an increase in ODC activity (Byus and Russell, 1974, 1975, 1976a,b; Hogan et al., 1974a; Mizoguchi et al., 1975; Reddy and Villee, 1975; Russell and Stambrook, 1975; Short et al., 1975; Zusman and Burrow, 1975; Canellakis and Theoharides, 1976; Costa et al., 1976; Oka and Perry, 1976; Russell et al., 1976a; Yamasaki and Ichihara, 1976; Byus et al., 1977, 1978a,b; Jungmann and Russell, 1977; Scheinman and Burrow, 1977; Costa, 1978a; Costa and Nye, 1978; Hochman et al., 1978; Insel and Fenno, 1978; Levine et al., 1978; Manen et al., 1978; Osterman and Hammond, 1978; Osterman et al., 1978; Rupniak and Paul, 1978; Klimpel et al., 1979; Rosenfeld and Barrieux, 1979; Russell and Haddox, 1979; Combest and Russell, 1980; Costa et al., 1980; Hibasami et al., 1980c; Madhubala and Reddy, 1980; Nichols and Prosser, 1980). However, there is no compelling evidence to indicate that involvement of cAMP is mandatory in the process of ODC induction, since in a large body of experimental results there was no relationship between the enhancement in ODC levels and the changes in cyclic AMP levels within the cell (Mangan et al., 1973; Thrower and Ord, 1974; Eloranta and Raina, 1975; Richman et al., 1975; Oka and Perry, 1976; Scalabrino and Ferioli, 1976; Collawn and Baggett, 1977; Jefferson and Pegg, 1977; Johnson and Sashida, 1977; Mufson et al., 1977; Chadwick et al., 1978; Insel and Fenno, 1978; Lau and Slotkin, 1979; Marks et al., 1979; Piik et al., 1979; Veldhuis and Hammond, 1979; Veldhuis et al., 1979; Combest and Russell, 1980; Gpyaho, 1980; Klingensmith et al., 1980). The rise in cAMP level and the increased ODC activity within the cell could be merely parallel manifes-

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tations of the interaction of the hormone with the cell. Therefore, although CAMP may increase ODC activity in some experimental systems, generalizations regarding the mandatory intracellular regulation of ODC activity by this cyclic nucleotide appear to be premature and not sufficiently grounded, until several additional normal and pathological tissues in many different growth states have been studied. The control of ODC activity by different hormones in different mammalian tissues has been extensively reviewed (Morris and Fillingame, 1974; Raina and J h n e , 1975; C. W. Tabor and Tabor, 1976; Janneet al., 1978). 2. Generally speaking, ODC has a fast rate of turnover in mammalian cells, as demonstrated by measuring its tile after inducing it and then measuring the rate of loss of its activity after inhibiting protein synthesis. In many mammalian tissues, the apparent half-life of ODC, determined after using different procedures for inducing the enzyme, is 10-30 min which is one of the shortest molecular turnover times among mammalian enzymes (Bachrach, 1973; Schimke, 1973; Janne et al., 1976; C. W. Tabor and Tabor, 1976). Interestingly, using rat liver ODC antiserum and an immunotitration method, Obenrader and Prouty (197%) have demonstrated that in rat liver the tllz for antigen is longer than that for enzyme activity loss by approximately 9 min. This result is in keeping with the hypothesis that the ODC molecule is inactivated prior to being degraded. However, a large number of studies both in uitro and in uiuo have demonstrated that the half-life of ODC may not be an absolute and constant value, valid for every tissue and every growth rate condition. In fact, in different organs of the rat or using a variety of mammalian cell cultures (normal or neoplastic), isolated cells, perfused organs (e.g., rat liver), lower eukaryotes, and even enucleated cells, a rather wide range of half-lives for ODC has been reported, making it evident that the half-life of ODC is influenced by a number of biological parameters, such as the growth conditions, the composition of the growth media, the growth rate of the cells, and even the type of inhibitor of protein synthesis used (Russell and Snyder, 1969; Russsll et al., 1970; Hannonen et at., 1972; Kay et al., 1972; Melvin et al., 1972; Aisbitt and Barry, 1973; Kay and Lindsay, 1973a; Mitchell and Rusch, 1973; Clark, 1974; Hogan and Murden, 1974; Hogan et al., 197413; Janne and Holtta, 1974; Lembach, 1974; O’Brien et al., 1975a; Bachrach, 1976a; Heller et al., 1976b; Prouty, 1976; Yamasaki and Ichihara, 1976; Chen and Canellakis, 1977; Clark and Greenspan, 1977,1979; Conroy et al., 1977; Jefferson and Pegg, 1977; Kallio et al., 1977a; Obenrader and Prouty, 1977b; Canellakis et al., 1978; McCormick, 1978a; Minaga et al., 1978; Poso et al., 1978; Pegg and McGill, 1979; Veldhuis et al., 1979; Lau and Slotkin, 1980). Gen-

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erally speaking, one can conclude that there is good evidence that in cell cultures the half-life of ODC is a variable function, whereas in in vivo systems the tllzfor the enzyme is relatively more constant. There is evidence that ODC exists in different molecular forms, since multiple forms of ODC have been separated from regenerating or hepatocarcinogen-treated rat livers, from different kinds of growing cell cultures, from lower eukaryotes, and from bacteria. In exponentially growing mouse fibroblasts, two forms of ODC have been found that differ with respect to pyridoxal5’-phosphate affinity but that are without appreciable difference in their K, for ornithine and that have similar activity half-lives (Clark and Fuller, 1976a; Fuller et al., 1978). Two separable forms of ODC with different K, values for ornithine but identical antigenic properties and molecular sizes have been isolated from livers of rats injected with thioacetamide and from regenerating rat liver (Prouty and Obenrader, 1976; Obenrader and Prouty, 1977a). The increases observed in heart ODC activity of mature rats after T3or isoproterenol administration or the innately higher ODC of 2-day-old neonates always involve a form of the enzyme with an increased affinity for substrate (Lau and Slotkin, 1979). In addition, the developmental decline of heart ODC activity in the rat can be explained by the disappearance of the high-affinity kinetic form of the enzyme (Lau and Slotkin, 1980).In a primitive eukaryote (Physarurn polycephalurn) the ODC activity consists of two interconvertible forms, which differ in their K, for pyridoxal phosphate and their molecular size, though their K , for the substrate, ornithine, is unchanged. In this case these forms appear to be two distinct states, i.e., active and less active, of a common enzyme protein (Mitchell, 1974, 1975; Mitchell and Sedory, 1974; Mitchell et al., 1976, 1978b; Mitchell and Carter, 1977; Sedory and Mitchell, 1977; Mitchell and Kottas, 1979). Escherichia coli also contains two ODCs: a “biosynthetic” enzyme found during normal exponential growth and a “biodegradative” one induced by growth at low pH in culture media enriched with amino acids. They are distinct but very similar proteins, with similar molecular weight and similar kinetic properties, but the antibody to the purified biodegradative ODC does not cross-react with the biosynthetic enzyme (Morris and Pardee, 1965,1966; Applebaum et al., 1975,1977). Moreover, the K, values for ornithine have been demonstrated to be significantly different in uninfected HeLa cells than in vaccinia virus-infected cells, being higher in the latter than in the former (Hodgson and Williamson, 1975). Besides the above-reported existence of multiple ODC forms in a given tissue or cell, there is also the possibility that ODC from different tissues in the same animal species can display different enzymic prop-

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erties. Comparative kinetic studies of ODC activity in brain and liver of the rat indicate that the brain ODC has a higher affinity for the substrate than the liver ODC (Butler and Schanberg, 1976). The exact relationship between the various forms of ODC in different tissues has not yet been completely elucidated. It is not yet known whether multiple enzyme forms represent different transcriptional products (i.e., isozymes) or posttranslational modifications (e.g., proteolytic cleavage). Nevertheless, however these questions are answered in the future, it seems to us important to point out the following: (1) it is not by chance that these multiple forms of ODC have been clearly identified and characterized in tissues and cells which were stimulated to growth by various stimuli or, at least, in which the ODC activity was increased or decreased by suitable variations of the media’s osmolarity; (2) thus, in 3T3 fibroblasts stimulated to growth, in hepatic hyperplasia either surgically or chemically induced, in Physarum polycephalum after osmotic shock, and in vaccinia virusinfected HeLa cells, the change in the growth state and the subsequent increase in ODC activity are generally accompanied by a predominant presence of those forms of ODC which show higher affinity for the substrate and/or for the coenzyme; (3) consequently, the possibility that the K, of the ODCs for the coenzyme and/or for the substrate might change represents, along with a concomitant change in the enzyme’s half-life, one of the key mechanisms available to the cell to fit ODC activity to the cell growth state and to environmental conditions. In fact, several lines of experimental evidence obtained in cells in culture suggest that frequently the increase in the half-life of a rapidly turning-over enzyme such as ODC represents an important, albeit not the only available, mechanism for the increase in cellular enzyme activity. However, conflicting and quite opposite results have been reported in this regard (Clark, 1974). Furthermore, it is important that in normal rat liver the ODC activity declined exponentially after treatment with cycloheximide, with a half-life rather similar to that observed in regenerating liver, in which there is intense stimulation of the enzyme activity in response to partial hepatectomy (Russell and Snyder, 1969). In conclusion, for the biological significance of the multiple forms of ODC, as for the half-life of ODC and for mediation by CAMP of ODC induction, no general rule can yet be drawn. Specific ODC antibodies have been prepared in different laboratories with purified enzyme preparations (Friedman et al., 1972; Holtta, 1975; Canellakis and Theoharides, 1976; Theoharides and Canellakis, 1976; Kallio et al., 1977c; Piik et al., 1977; Scheinman et al., 1977;

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Kallio, 1978; Poso et al., 1978). By immunological techniques it was possible to ascertain that the rise in ODC activity in some organs, such as liver or thyroid, of the rat after in vivo or in vitro induction of the enzyme by hormones (GH or TSH) is accompanied by increased amounts of immunoprecipitable protein, which indicates de novo synthesis of the enzyme protein (Holtta, 1975; Scheinman et al., 1977). This result has been confirmed in cultured neoplastic cells (Canellakis and Theoharides, 1976). Conversely, the decrease in ODC activity in the regenerating liver or the ventral prostate of the rat after in vivo injections of inhibitors (such as putrescine, 1,3-diaminopropane, and cycloheximide) were usually, if not always, associated with a similar and parallel decay in the amount of immunoreactive protein, as revealed by immunotitration of the enzyme (Kallio et al., 1977c; Piik et al., 1977; Kallio, 1978; Poso et aZ., 1978). The different preparations of antiserum to rat liver ODC showed no cross-reactivity with ODC derived from E. coli (Theoharides and Canellakis, 1976). Finally, the induced ODC was identical with the noninduced one, as demonstrated by titration with an antibody monospecific for this enzyme and by heat stability (Canellakis and Theoharides, 1976). ODC activity itself has been suggested to be a regulatory factor for RNA polymerase I activity and is even considered to be a trigger for the stimulation of RNA polymerases (Manen and Russell, 1975a,b, 1976, 1977a,b; Russell et al., 1976a; Manen et al., 1977; Haddox and Russell, 1980). However, several results with different biological systems have indicated that this is not always so (Spaulding, 1977; Ferioli et al., 1980). Particularly, it should be mentioned that in postischemic rat liver the activation of ODC can be abolished without impairing the activation of RNA synthesis that occurs in this experimental system (Ferioli et aZ., 1980). Therefore, one can conclude that, if there is a link between ODC and RNA metabolism, it is by no means a necessary one. Without any doubt, the regulation of ODC activity in eukaryotic cells represents at the present one of the most intriguing questions in the control of polyamine biosynthesis. This problem is also of paramount importance because ODC catalyzes the rate-limiting step in the polyamine biosynthetic pathway. Unlike ODC from E. coli, in which this enzyme appears to be largely under the regulation of certain guanine nucleotides (Holtta et al., 1972, 1974; Sakai and Cohen, 1976a,b), mammalian ODC is controlled not by low-molecular-weight effectors (Pegg and Williams-Ashman, 1968a; Janne and WilliamsAshman, 1971b; Friedman et al., 1972; Clark, 1974) but by several distinctly different mechanisms. Mammalian ODC does not exhibit

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allosteric behavior, since it is not at all directly modulated by small biomolecules, with the exception of thiols. Experiments carried out in the assay systems in vitro and aimed at demonstrating direct effects of the product of the ODC reaction (i.e., putrescine) and of the subsequent polyamines (spermidine and spermine) on the decarboxylase have shown that these molecules are only very weak direct competitive inhibitors of catalytic activity of ODC (Pegg and WilliamsAshman, 1968a; Raina and Janne, 1968; Morris et al., 1970; Janne and Williams-Ashman, 1971b; Ono et al., 1972; Clark, 1974; Morley and Ho, 1976; Pegg and McGill, 1979). By contrast, using in vivo and in vitro biological living systems (i.e., intact animals, cultured cells, and even cytoplasts) exogenous polyamines, particularly putrescine, are very effective in depressing ODC activity and in preventing the rise in ODC activity caused by several means. It has been observed, as well, that administration of certain other diamines not normally present in living organisms decreases ODC activity in various rat tissues in vivo and in cultured cells (Schrock et al., 1970; Kay and Lindsay, 1973b; Janne and Holtta, 1974; Clark and Fuller, 1975, 1976b; Canellakis and Theoharides, 1976; Fong et al., 1976; Heller et al., 1976b, 1977b, 1978; Poso and Janne, 1976a,b; Friedman et al., 1977a; Guha and Janne, 1977; Jefferson and Pegg, 1977; Kallio et al., 1977a-d; McCann et al., 1977; Piik et al., 1977; Poso, 1977; Poso et al., 1977; Kallio, 1978; Mitchell et al., 1978a; Pegg et al., 1978; Bethel1 and Pegg, 1979a; Clark and Greenspan, 1979; Grillo et al., 1980; Klingensmith et al., 1980; Stoscheck et al., 1980). The mechanisms by which exogenous polyamines and synthetic diamines suppress the expression of ODC activity (i.e., the so-called amine repression of ODC) are not well understood. In summary, for such a regulation of ODC activity, two main different, though not conflicting, explanations have been proposed. Evidence has been presented that this inhibitory effect on ODC activity of di- and polyamines may be mediated through a decline in the synthesis of the enzyme at some posttranscriptional steps. The rapidity of the amine action, which is comparable with that of cycloheximide and other inhibitors of eukaryotic protein synthesis, is consistent with this hypothesis (Janne and Holtta, 1974; Clark and Fuller, 1975; Kallio et al., 1977a,c).Furthermore, some evidence supports the idea that the action of the diamines might involve transcriptional control elements of gene expression (Janne and Holtta, 1974; Kallio et al., 1977a). Thus, the inhibitory action of putrescine on ODC activity is not a simple direct feedback inhibition. In any case, this decrease in ODC activity by

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putrescine appears to be due to the diamine per se rather than to a subsequent polyamine, i.e., spermidine or spermine (Pegg et al., 1978). In fact, in vivo treatment of rats with MGBG, which prevents spermidine synthesis by an inhibition of SAMD activity, did not prevent the putrescine-induced decrease in rat liver ODC activity (Pegg et al., 1978). Further, some nonphysiological diamines, structural analogs of putrescine that are poor substrates for the propylamine transferase reaction in which spermidine is formed and that are thus converted into polyamine analogs to a very limited extent, were also able to decrease ODC activity in vivo (Pegget al., 1978). However, the picture is further complicated by the finding that the reduction in ODC activity of the primitive eukaryote Physarum polycephalum brought about by putrescine, spermidine, or spermine is due not to a decrease in enzyme molecules but rather to the rapid conversion of the active enzyme to a stable, catalytically less active form (Mitchell et al., 1978a). An alternative, fundamentally different explanation of the inhibitory effects of di- and polyamines on ODC activity in vivo has been recently put forward by Canellakis and his co-workers. These authors found that the addition of putrescine to cell cultures or the injection of partially hepatectomized rats with putrescine swiftly induced in the cell or in the liver the formation of a nondiffusible and noncompetitive inhibitor of ODC activity, which they called “ODC antizyme” (Fong et al., 1976; Heller et al., 1976a,b, 1977a,b, 1978; Heller and Canellakis, 1980).The production of this ODC antizyme has been confirmed in other biological systems and tissues by other authors (Friedman et al., 1977a; Jefferson and Pegg, 1977; Kallio et al., 1977c, 1979; McCann et al., 1977; Minaga et al., 1978; Pegg et al., 1978; Grillo et al., 1980; Klingensmith et al., 1980; Stoscheck et al., 1980; Weekes et al., 1980), with very few negative reports (Clark and Fuller, 1976b; Mitchell and Carter, 1977; Piik et al., 1977; Mitchell et al., 1978a; Pegg et al., 1978; Weekes et al., 1980). Certainly, the discovery of an ODC antizyme is a very important addition to the understanding of molecular regulation of the biosynthesis of polyamines. The antizyme appears to be a protein, since it is sensitive to proteinase but not to nucleases, and its induction by putrescine is inhibited by cycloheximide but not by actinomycin D, indicating that synthesis of protein is occurring on stable mRNA templates. The mechanism of action of this macromolecular inhibitor seems to involve reversible binding of the inhibitor to ODC, resulting in a loss of catalytic activity of the enzyme and disappearance of the free inhibitor. Interestingly, eukaryote ODC activity is also inhibited by a negative effector of E.

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coli ODC, which, although it has been isolated from this bacterial strain, shows characteristics similar to those of the ODC antizyme present in eukaryotic cells (Kyriakidis et al., 1978). A positive effector of ODC activity has also been isolated in E . coli (Kyriakidis et al., 1978). Finally, it is worthwhile to mention that in mammalian cells the ODC antizyme is a normal component, present at least in the livers of rats untreated with putrescine or other diamines (Heller et al., 1977b). In uninduced rat liver cells the largest part of the ODC antizyme activity is localized in the nuclei (in the nucleolus as well as in the nucleoplasm), the remaining activity being found in some subcellular particles, i.e., in the smooth endoplasmic reticulum and in the rough endoplasmic reticulum (Heller et al., 197713). Regardless of the mechanisms that may be involved in regulating the ODC activity in eukaryotic cells by physiological polyamines, putrescine certainly has a peculiar and prominent role in comparison with spermidine and spermine in such a regulation. In fact, putrescine may regulate the activities of both ODC and SAMD in eukaryotes, since this natural diamine sharply depresses ODC activity, on the one hand, and, on the other hand, markedly stimulates SAMD (see Section I,B,2). Surprisingly, it has been observed that, in general, the ODC of most cells is sensitive to external polyamine concentrations several orders of magnitude lower than that found internally. This greater sensitivity of mammalian ODC to exogenous polyamines, despite the presence of much higher endogenous levels of polyamines, hints at the possibility that the mechanism controlling ODC activity may be regulated by a polyamine-sensitive site on the external cell membrane (Canellakis et al., 1978). Although there is, as yet, no conclusive evidence that extracellular di- and polyamines can regulate ODC activity at such a site, particularly because these amines have been demonstrated to be taken up into the cell by an active-transport system (Pohjanpelto, 1973, 1976; Lajtha and Sershen, 1974; Pateman and Shaw, 1975; Kano and Oka, 1976), some experimental evidence exists for the presence of membrane-associated sites that affect ODC activity (Quash e t al., 1971, 1972, 1976, 1978; Richman et al., 1975; Chen et al., 1976a; Canellakis et al., 1978; Gibbs et al., 1980). In fact, agents known to affect the membrane via the cytoskeleton (such as colchicine, cytochalasin, and vinblastine) inhibit the induction of ODC in vivo and in cultured cells (Richman et al., 1975; Chen et al., 1976a), and, furthermore, putrescine has been proved to be associated with sites on the surface of different types of cells (Quash et al., 1971, 1972, 1976). The regulation of cellular ODC activity is not limited to the different

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intracellular control mechanisms, such as those discussed previously (i.e., RNA synthesis, antizyme induction, changes in the enzyme's half-life, changes in the molecular form of the enzyme, translational control exerted by polyamines). Extensive studies indicate that many environmental parameters, including the ion content of the media, can profoundly influence the levels of ODC activity in different kinds of tissues. More exactly, decreasing the osmolarity of the culture medium results in marked stimulation of ODC activity, whereas hyperosmotic culture medium prevents an increase in ODC activity inside the cells (Munro et al., 1975; Friedman et al., 197713; Mitchell and Kottas, 1979; Perry and Oka, 1980). Furthermore, high external levels of certain cations (Ca", Na+, K+, and M$+) inhibit ODC activity (Chen et al., 1976b; Otani et al., 1980), whereas high extracellular Ca2+levels are strictly required for both of the separate pathways of ODC induction, i.e., the cAMP dependent and the cAMP independent (D'Amore and Shepro, 1978; Gibbs et al., 1980). It seems to be of great interest to note (a) that there is a very close relationship between some inorganic cations and the organic polyamine polycations in regulating ODC activity and (b) that inorganic cations, with the notable exception of calcium, seem to act in controlling ODC activity like organic polyamine polycations, since changes in the pools of the cations cause inverse responses in the ODC activity. The reader can obtain further and more detailed information about the different mechanisms involved in the control of ODC activity in mammalian cells from the excellent reviews provided by Canellakis et al. (1979), Bachrach (1980), and McCann (1980). 2. S-Adenosyl-LMethionine Decarboxylase In most organisms, S-adenosyl-L-methionine decarboxylase (SAMD) (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) plays a pivotal role in polyamine biosynthesis by contributing S-methyladenosylhomocysteamine (i.e., decarboxylated S-adenosyl-L-methionine), which in turn donates its propylamine moiety to putrescine to give spermidine and then to spermidine to give spermine. Contrary to earlier reports (Pegg and Williams-Ashman, 1968b, 1969; Feldman et al., 1971, 1972; Manen and Russell, 1974), it is now well established that the decarboxylation of S-adenosyl-L-methionine and the propylamine transfer reaction are catalyzed by different enzymes (Coppoc et d., 1971; Janne and Williams-Ashman, 1971a; Janne et al., 1971a,b; Pegg, 1974; Sturman, 1976). Three types of SAMD activities with different in vitro biochemical features have

176

GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

been described: (1)prokaryotic enzymes, which are Mgz+dependent but putrescine insensitive (Wickner et al., 1970; Coppoc et al., 1971; Poso et al., 1976); (2) enzymes of lower eukaryotes, which are not influenced either by divalent cations or by putrescine (Mitchell and Rusch, 1973; Poso et al., 1975a,b, 1976); and (3) the enzymes from higher organisms and from yeasts, which are stimulated markedly by putrescine (also at physiological concentrations of this diamine) and, to a lesser extent, by spermidine and cadaverine in the assay system (Coppocet al., 1971; Janne and Williams-Ashman, 1971a; Janne et al., 1971a; Zappia et al., 1972; Poso et al., 1975a, 1976; Sakai et al., 1979; Wilson et al., 1979). The important effect of putrescine on the activity of mammalian SAMDs must be connected with the remarkable decrease caused by the diamine in the apparent K,,, value of the enzyme for S-adenosyl-L-methionine (Hannonen, 1975; Poso et al., 1975a; Oka et aZ., 1978b; Sakai et aZ., 1979). In the presence of a saturating concentration of the activator putrescine, SAMD has apparent K , values for adenosylmethionine ranging from 0.09 mM to 0.02 mM, depending on the tissue source of the enzyme (Coppoc et al., 1971; Hannonen, 1976; Porta et al., 1977; Oka et al., 197813). It is therefore likely that the activity of SAMD in vivo can be significantly modulated by changes in the amount of putrescine present inside of the cell, as consequences of corresponding changes in ODC activity. Amazingly enough, however, Sakai et al. (1980) recently demonstrated that the increases in the intracellular levels of putrescine, induced in cultured mouse mammary tissue by different means, resulted in a decrease of both the activity and the amount of SAMD in tissue. At present, this dissociation between the effects of putrescine on SAMD in the assay system and in cells remains to be explained. The absence of putrescine activation of prokaryotic and some lower eukaryotic SAMDs can conceivably be attributed to the near absence of spermine and its synthase in these species (Poso et al., 1976). Analogously, the stimulation by putrescine of eukaryotic SAMD has evolutionary significance, since this property presumably enables the cell to have adequate amounts of decarboxylated S-adenosyl-L-methionine for polyamine biosynthesis. Another unique characteristic of SAMD isolated from eukaryotic sources is the strong in vitro inhibition of the enzyme by S -methylhomocystearnine, i.e., by the product of the reaction (Yamanoha and Samejima, 1980).The presence of such an inhibition is another difference between SAMD from prokaryotic and SAMD from eukaryotic organisms (Raina and Janne, 1975).

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Like ODC, mammalian SAMD is an enzyme located in both the cytosol fraction (Schmidt and Cantoni, 1973; Sturman, 1976; Symonds and Brosnan, 1977; Wilson et al., 1979) and the nucleus of the cell (McCormick, 1977, 1978a). It has been purified from different organs of the rat and from baker’s yeast and its molecular weight determined. Like ODC, mammalian SAMD has a short biological half-life, which ranges between 20 and 120 min, depending on the tissue studied (Russell and Taylor, 1971; Hannonen et al., 1972; Russell and Potyraj, 1972; Fillingame and Morris, 1973; Kay and Lindsay, 1973a; Mitchell and Rusch, 1973; Pegg et al., 1973; Janne and Holtta, 1974; Pegg and Jefferson, 1974; O’Brien et al., 1975a; Eloranta et al., 1976a; Janne et al., 1976; Jefferson and Pegg, 1977; Grillo et al., 1978a; Poso et al., 1978; Pegg, 1979). We have no convincing evidence thus far for the existence of multiple forms of SAMD in mammalian cells. Although earlier works suggested that pyridoxal 5-phosphate is a cofactor for eukaryotic SAMD (Feldman et al., 1972; Sturman and Kremzner, 1974; Hannonen, 1975), later studies have demonstrated that, despite other differences, the putrescine-activated SAMD (e.g., that of yeasts and of rat liver) resembles the prokaryote SAMD (e.g., that of E. coli) in having a covalently bound pyruvate cofactor (Wickner et al., 1970; Cohn et al., 1977; Pegg, 1977a; Demetriou et al., 1978). Pertinent to these findings are several reports indicating that hepatic SAMD activity is not at all affected in rats fed on a diet deficient in vitamin Be (Eloranta et al., 197613; Hannonen, 1976; Pegg, 197%). The substrate specificity of SAMD from eukaryotic sources is quite rigorous, although some analogs of S-adenosyl-L-methionine containing selenium have been shown to be attacked by the enzyme (Pegg, 1969; Zappia et al., 1972). Like ODC, mammalian SAMD activity is influenced by the nutritional state (Eloranta and Raina, 1977) and shows circadian rhythm in several organs of the rat (Scalabrino et al., 1979a). The SAMD activity can increase in mammalian tissues (both in vivo and in cultured cells) in response to various stimuli for cell growth or differentiation, such as partial hepatectomy or some anabolic hormones, so that SAMD is also an inducible enzyme (Kaye et al., 1971; Russell and Lombardini, 1971; Russell and Taylor, 1971; Hannonen et al., 1972; Oka and Perry, 1974a; Eloranta and Raina, 1977; Feil et al., 1977; Sakai et al., 1977, 1978; Igarashi et al., 1978). However, the observed enhancement of SAMD levels after these stimuli is usually smaller than that observed in ODC activity under the same experi-

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

mental conditions. Moreover, although ODC and SAMD activities are frequently enhanced in a coordinated way in the tissues in response to stimuli, in many tissues large increases in ODC activity are often not followed by proportionate rises in SAMD activity (Williams-Ashman et al., 1977; Williams-Ashman and Canellakis, 1979; Ferioli et al., 1980). Highly purified specific antibodies to SAMD from the livers of rat and mouse have been prepared (Pegg, 1979; Sakai et al., 1979),and by immunotitration it was shown that the increase in SAMD activity in the tissues during regenerative growth or after appropriate hormonal stimulation is due to an increase in the amount of enzyme protein (Pegg, 1979). It was also found that, after treatment of the rats with cycloheximide, the antigen disappeared, with a tllzslightly longer than that for the enzyme activity (Pegg, 1979). These data for SAMD are quite similar to those for ODC and may mean that, as in the case of ODC, the enzyme is first inactivated and then degraded. Convincing evidence in vivo or in cultured cells for the regulation of SAMD activity by di- and polyamines, comparable to the so-called amine repression of ODC activity, is for the moment lacking. In fact, either quite opposite or ambiguous findings have been reported about the effects of in vivo administration of putrescine or spermidine on SAMD levels in different organs of the rat. An in viuo inhibitory action of spermidine on the hepatic and muscular enzymes was found by some authors (Janne and Holtta, 1974; Hopkins and Manchester, 1980), and this inhibition was also confirmed in cultured lymphocytes (Kay and Lindsay, 1973b). However, in vivo stimulation by spermidine of hepatic SAMD in the chicken has been reported (Grillo et al., 1978a). Furthermore, putrescine was found to not affect or to scarcely stimulate SAMD levels in liver of rats or chickens when given intraperitoneally (Janne and Holtta, 1974; Grillo et al., 1978a) or when added to cultured lymphocytes (Kay and Lindsay, 1973b). The injection of the same diamine or of 173-diaminopropanebrought about a decrease in the activity of this decarboxylase in the seminal vesicle but not in the prostate of the rat (Piik et al., 1977). As recently demonstrated by Zappia and his co-workers, decarboxylated SAM is also the precursor of some of the newly identified polyamines, i.e., sym-norspermidine and sym-norspermine. Two molecules of this decarboxylated SAM are required for the biosynthesis of s ym-norspermidine and three molecules for the biosynthesis of symnorspermine (De Rosa et al., 1978; Zappia et al., 197913). The peculiarity of the biosynthetic pathway of these “new polyamines” lies in the fact that S-5’-deoxyadenosyl-(5’)-3-methylthiopropylamine, i.e., decarboxylated SAM , contains the entire carbon skeleton of the mole-

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cules of sym-norspermidine and sym-norspermine, which are also called caldine and thermine.

3. Spermidine Synthase This enzyme (S-methyladenosylhomocysteamine:putrescine aminopropyltransferase, EC 2.5.1.16) catalyzes the transfer of the propylamine moiety of decarboxylated SAM to putrescine to form spermidine, thiomethyladenosine, and one proton. When measured under optimal assay conditions in soluble extracts of most eukaryotic tissues examined so far, the activity of this synthase is noticeably higher than the activities of the two polyamine biosynthetic decarboxylases (Raina and Janne, 1975). Spermidine synthase from eukaryotic sources has not been fully purified and characterized. No cofactor requirement has been reported for this aminopropyltransferase. The K , value of spermidine synthase for synthetic decarboxylated SAM has been found to be below 4 p M (Samejima and Nakazawa, 1980), and a similar value was determined with naturally occurring decarboxylated SAM (Raina and Janne, 1975). Unlike the aminopropyltransferase of prokaryotes, for which not only putrescine but also cadaverine and spermidine can act as acceptors in the aminopropyl transfer reaction (Bowman et al., 1973), mammalian spermidine synthase can use putrescine and, to a slight degree, l74-diarnino-2-butene, but not spermidine or 1,3-diaminopropane7as acceptors of the aminopropyl group from decarboxylated SAM (Samejima and Nakazawa, 1980). Conflicting reports are available as to whether 1,Sdiaminopentane (cadaverine) can be a substrate for spermidine synthase from eukaryotic organisms (Kallio et aZ., 1977d; Hibasami and Pegg, 1978a; Samejima and Nakazawa, 1980). Therefore, we can conclude that the mammalian enzyme is quite specific for the formation of spermidine. Moreover, the mammalian spermidine synthase transfers only the aminopropyl group, not the aminoethyl or aminobutyl groups (Samejima and Nakazawa, 1980). Unlike the two polyamine bios ynthetic decarboxylases, spermidine synthase has a rather long apparent biological half-life (Haina and Janne, 1975; Oka et al., 1977). This enzyme is markedly inhibited in vitro by SAM, by its synthetic or natural analogs and derivatives, and by cadaverine (1,5-diaminopentane) (Hibasami and Pegg, 197813; Hibasami et al., 1980b). Spermidine synthase activity can be increased by hormones in an inducible manner (Oka et al., 1977; Kapyaho et al., 1980). Interestingly, in some prokaryotes that have no detectable SAMD

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

activity, a novel pathway of spermidine synthesis has been described, in which spermine synthase is absent (Tait, 1976). According to the proposed scheme, L-aspartic-P-semialdehyde condenses with putrescine to yield a Schiff base, which is then reduced to carboxyspermidine by an NADPH-dependent step. Carboxyspermidine, in turn, undergoes a pyridoxal 5-phosphate-dependent enzymatic decarboxylation to give rise to spermidine. Of further relevance to this is the recent demonstration of the coexistence of the two pathways, i.e., the classical (which includes SAMD and spermidine synthase) and the new one (in which P-aspartylsemialdehyde is precursor), for spermidine biosynthesis in Lathyrus sativus seedlings (Srivenugopal and Adiga, 1980). 4. Spemnine Synthase

This enzyme (S-methyladenosylhomocysteamine:spermidine aminopropyltransferase) catalyzes the synthesis of spermine, using decarboxylated SAM as the donor of the propylamine group and spermidine as the acceptor. Other products of the reaction are 5-methylthioadenosine and one proton. This enzyme has been highly purified and characterized and shows a high affinity for S -methyladenosylhomocysteamine,with a K, value of about 0.6 p M (Pajula et al., 1978, 1979; Pajula and Raina, 1979). Purified spermine synthase is inhibited by putrescine and even more by one of the reaction products, methylthioadenosine (Pajula and Raina, 1979; Hibasami et aZ., 1980b). Putrescine was found to be a competitive inhibitor of spermine synthesis (Pegg and WilliamsAshman, 1970; Hannonen et al., 1972). SAM is also a strong in vitro inhibitor of the activity of the enzyme, which is much more depressed by this compound than is spermidine synthase (Hibasami et al., 1980a,b). Another physiological in vitro inhibitor of spermine synthase is 175-diaminopentane (Hibasami and Pegg, 1978b). No cofactor or coenzyme appears to be needed for the synthesis of spermine by spermine synthase. This enzyme is also inducible by hormones (Gpyaho et al., 1980). However, we do not know at present whether the in vitro inhibition of the aminopropyltransferases by SAM, decarboxylated SAM, cadaverine, or 5’-methylthioadenosine also occurs in vivo, because the concentrations of SAM in the tissues are low (Eloranta et al., 1976b; Eloranta, 1977, 1979; Eloranta and Raina, 1977; Hoffman et aZ., 1979) and those of decarboxylated SAM (Hibasami et al., 1980d) and cadaverine are very low. Very few measurements of cellular 5’-methylthioadenosine content have been made (Rhodes and WilliamsAshman, 1964; Seidenfeld et aZ., 1980).

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181

C. S -ADENOSYL-L-METHIONINE S-Adenosyl-L-methionine (SAM) has become increasingly important in cell biochemistry. In fact, SAM links two biochemically important processes: biological methylations and polyamine biosynthesis. SAM is employed as a methyl donor in some, but by no means all, enzymic methyltransferase reactions. Additionally, t w o recently found roles for SAM appear to be of particular importance: (1) SAM is required for both bacterial chemotaxis and the chemotaxis of human monocytes (Aswad and Koshland, 1974,1975; Springer and Koshland, 1977; Pike et al., 1978); these findings support a relationship between SAMmediated transmethylation reactions and immune and inflammatory functions; (2) SAM is required, with ATP and magnesium ions, by the class I type of bacterial restriction enzymes (Linn et al., 1977; Cat0 and Burdon, 1979; Smith, 1979). SAM is also of paramount importance in the normal and pathological biochemistry of the mammalian central nervous system [e.g., it is involved in the methylation of some neurotransmitters (Wurtman, 1979), and its abnormal metabolism was proposed to be connected with some psychotic disorders (Baldessarini et al., 1979)] and in preventing, or at least reducing, the severe cell hepatic toxic injury induced by several drugs and molecules (Stramentinoli et al., 1978b, 1979). Just as for the three major polyamines, an active transport system with high affinity for SAM has been described in mammalian cells (Mizoguchi et al., 1972; Pezzoli et al., 1978; Stramentinoli et al., 1978a; Zappia et al., 1978a). Finally, at least one rate-limiting factor of SAM synthesis in mammalian tissues seems to be the tissue concentration of methionine available to the cell (Eloranta, 1977, 1979). The pathways of SAM metabolism and catabolism and the interconnections between transmethylation and transsulfuration reactions, on the one hand, and polyamine synthesis, on the other hand, are presented in Fig. 3. For further details about the different roles played by SAM in the biochemistry of eukaryotic and prokaryotic cells, we refer the reader to two books on these topics (Salvatore et al., 1977; Zappia et al., 1979a).

D.

5’-METHYLTHIOADENOSINE

It is noteworthy that concomitant with the reactions of both spermidine and spermine synthases there is stoichiometric production of 5‘-methylthioadenosine (5’-MTA). In microorganisms this nucleoside is synthesized through several different pathways from the common

182

GIUSEPPE SCALABFUNO AND MARIA E. FEFUOLI

L - METHIONINE METHYL1 HIOADENOSINE

P; + PP;

0

PUTRESCINE S-METHYLADENOSVL HOMOCYSTEAMINE

A-ACCEPTORS

S-ADENOSYL-1- HOMOCYSTEINE ~

@

A T P c AMP+

II

IMP+

ADENOSINE

L - HOMOCYSTE INE

INOSINE

L - CYSTATHI0 NINE

HVPOXANTHINE

@

\I/

L-HOMOSEAINE + L-CYS ElNE

$~o+zo~

WJ2

CoA

URIC ACID

TAURINE

-TL-cvsT ZH

GLUTAT HI ONE

0 METHIONINE ADENOSYLTRANSFERASE (EC 2 5 1 6 ) @ S-ADENOSYL-L-METHIONINE DECARBOXYLASE (EC 4 1 1 5 0 ) 0 METHYLTRANSFERASES @

@ @

@ @ @ @ @ @ @

S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE (EC 3 3 1 1 ) ADENOSINE DEAMINASE ( E C 3 5 4 17) INOSINE -GUANOSINE PHOSPHORYLASE ( E C 2 4 2 1 ) XANTHINE

OXIDASE (EC 1 2 3 2 )

ADENOSINE KINASE

(EC 2 7 1 2 0 )

HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE (EC 2 4 2 8 ) N5-METHYL FH4 HOMOCYSTEINE METHYLTRANSFERASE BETAINE

L-HOMOCYSTEINE-S-METHYLTRANSFERASE (EC 2 1 1 5)

CYSTATHIONINE

0-SYNTHASE

(EC 4 2 1 2 2 )

CYSTATHIONINE T-LYASE (CYSTATHIONASE) (EC 4 4 1 1)

FIG. 3. Pathways of S-adenosyl-L-methionine synthesis and catabolism in mammalian tissues. Interconnections between the transmethylation and transsulfuration reactions are also shown.

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POLYAMINES IN MAMMALIAN TUMORS

precursor, SAM. Only two of these biosynthetic routes are operative in mammalian tissues. One reaction that leads to the formation of this thioether is the direct cleavage of SAM by a specific SAM lyase (or SAM-splitting enzyme) to form 5’-MTA and a-amino-y-butyrolactone. The second route is the transfer of a propylamine moiety from decarboxylated SAM to putrescine or spermidine to yield spermidine or spermine, respectively; this one is quantitatively more important. By this reaction, one molecule of 5’-MTA is produced per molecule of spermidine formed, and 2 molecules of 5’-MTAare produced per molecule of spermine formed (Ferro et d., 1976). Larger amounts of 5’-MTA are formed in thermophilic bacteria during the biosynthesis of some newly identified polyamines (e.g., sym-norspermidine and sym-norspermine), since two molecules of 5’-MTA are produced per molecule of sym-norspermidine and three molecules of 5’-MTA per molecule of sym-norspermine (De Rosa et al., 1978; Carteni-Farina e t al., 1979; Zappia et al., 1980). The pathways for 5’-MTA biosynthesis in mammalian cells are presented in Fig. 4. It has long been customary to regard 5’-MTA as a waste product of the last steps of the polyamine biosynthetic pathway. However, several more recent lines of experimental evidence have attributed important biological roles to 5’-MTA. In fact, 5’-MTA has been found to inhibit the proliferation of human lymphocytes stimulated to multiply by mitogens (Vandenbark et al., 1980) and to inhibit the incorporation SPERMIDINE

+

S’-METHYLTHIOADENOSINE]

PUTRESCINE

coq

SPERMlDlNE

DECARB~XYLATED

S - ADENOSY L-L-MET HlONlNE

,A SpERMlNE +

I5’-METHYLTHIDADENOSlNE

]

S -ADENOSYL - L‘- METHIDNINE

HOMOSEAINE LACTONE

I 5’-METHYLTHIOADENOSINE

0 S-ADENOSYL-L-METHIONINE-SPLITTING FIG.

ENZYME

4. Routes of biosynthesis of 5’-methylthioadenosine in mammalian cells.

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

of labeled uridine into RNA in explanted salivary glands from Drosophtla melanogaster (Law et al., 1976). On the other hand, 5’-MTA or some metabolite of it has been demonstrated to be required for the growth of certain cell lines in culture (Toohey, 1977, 1978). Moreover, the ubiquitous distribution and the high activity of the enzyme 5’-MTA phosphorylase (EC 2.4.2.1), which degrades this and adenine in mamcompound to 5’-methylthioribose-l-phosphate malian tissues (Cacciapuoti et al., 1978; Garbers, 1978; Zappia et al., 1978c, 1980; Ferro et al., 1979), accounts for the low intracellular levels of 5’-MTA in mammalian cells. Any accumulation of 5’-MTA would probably be deleterious to cells not only because of the adverse effect of this nucleoside on transmethylation but also because it would deplete the adenine nucleotide pool. However, although the metabolic fate of 5’-methylthioribose-l-phosphate, produced by enzymic cleavage of 5’-MTA, remains largely unknown, the adenine produced by the same metabolic reaction is available to reenter the pool of adenine nucleotides. Although 5‘-MTA is a strong in uitro inhibitor of spermine synthase (Pajula and Raina, 1979), the real inhibition of polyamine production displayed in uiuo by 5’-MTA may be only of little importance because of the rapid degradation of this nucleoside. Williams-Ashman et al. (1973, 1977), Williams-Ashman and Canellakis (1979), and Hibasami et al. (1980b) have suggested that the polyamine biosynthetic pathway may have as its major function the production of 5’-MTA rather than that of polyamines. However, partially clarifying this question, quite stimulating observations have been provided by Nicolette et al. (1980) and Seidenfeld et al. (1980). These authors demonstrated that, in both the ventral prostate and the uterus of the rat, 5’-MTA phosphorylase activity was markedly reduced by gonadectomy and, conversely, markedly and selectively enhanced by the administration of the appropriate sex hormones to the gonadectomized rats. Therefore, it is very interesting to notice that, at least in the prostate or uterus of the rat, 5’-MTA phosphorylase and the two biosynthetic decarboxylases show the same coordinated behavior in response to castration and stimulation by sex hormones, since the activities of all three enzymes are decreased by castration and increased by hormone administration (Seidenfeld et al., 1980). It is conceivable that the similar behaviors of these three enzymes are a synergistic and integrated response of the mammalian target tissues to hormonal stimulation, leading simultaneously to a stimulation of polyamine biosynthesis and to maintenance of low levels of 5‘-MTA inside the cell. In this case, the well-known stimulating effects

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of polyamines on cell growth and differentiation would be strengthened by a concomitant decrease in the inhibitory effect on cell growth of 5’-MTA. Therefore, although it is still premature to draw any final conclusion about the major function of the polyamine biosynthetic pathway, it is tempting to speculate that all of the final products of polyamine biosynthesis (i.e., polyamines and 5’-MTA)are of physiological impor: tance and that 5’-MTA phosphorylase may also act in a coordinate way with some enzymes involved in the biosynthesis of polyamines. Thus, the same biosynthetic route could give rise at the same time to compounds acting in quite opposite ways on cell growth: both stimulating compounds (i.e., polyamines) and inhibiting compounds (i.e., 5’MTA). This would allow the cell to delicately regulate its growth and differentiation under different biological situations by suitably adjusting the intracellular concentrations of these compounds.

E. CATABOLISM O F THE MAJORPOLYAMINES MAMMALIAN ORGANISMS

IN

Historically, one of the earliest demonstrated physiological roles of polyamines is the activity of these substances as growth factors for certain types of microorganisms (Herbst and Snell, 1948). However, only one year after the first report, it was found (Rozanski et al., 1949) that human semen inhibited the growth of certain bacteria. Although the exact mechanisms of this inhibition were not completely understood at once, it was established that the active principle for the inhibition was associated with the liquid part of the semen and not with the spermatozoa. Later, it became clear that one of the unique properties of human semen is its high content of the triamine and tetraamine (mostly spermine and, to a lesser extent, spermidine), in comparison with other body fluids and tissues, and its high diamine oxidase activity, which yields the cytotoxic oxidized derivatives of these polyamines. Therefore, in an interval of only one year, the theoretical and biochemical bases were laid that later would be fully developed to the conception of the polyamines and their oxidized derivatives as parts of an integrated biochemical system devoted to the regulation of cell proliferation and growth, by means of stimulating substances (i.e., polyamines) and inhibiting substances (i.e., oxidized derivatives of polyamines). The existence of a strict, functional connection between the polyamines and their catabolic products has been recently confkmed at the enzymological level by an interesting report in which

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GIUSEPPE SCALABFUNO A N D MAIUA E. FEFUOLI

diamine oxidase activity was shown to parallel ODC activity in rat small intestine mucosa (Baylin et al., 1978). Although there are a variety of amino oxidase activities in various animal tissues, the exact role played b y these enzymes in controlling tissue levels of polyamines remains unclear. At present, the classification of amine oxidases is quite confused. 1. Oxidation of Spemnidine and Spermine Amine oxidases are enzymes that are widely distributed in animal tissues and are classically divided into two distinct families: (1) monoamine oxidases [ MAO; amine : oxygen oxidoreductase (deaminating) (flavin-containing); E C 1.4.3.41, which act on primary, secondary, and tertiary amines and (2) diamine oxidases [DAO; diamine :oxygen oxidoreductase (deaminating) (pyridoxal-containing); E C 1.4.3.61, which act on aliphatic diamines, including histamine, and polyamines, and on primary monoamines. The identity of histaminase and diamine oxidase is generally accepted (Zeller, 1965). However, there are considerable differences in the substrate affinities of the DAO enzyme(s) between species and between tissues. The most quantitatively important catabolic pathway of polyamines is by oxidation. Putrescine, spermidine, and spermine are oxidized or cleaved by various enzymes from different sources: mammalian tissues, bacteria, plants, and such physiological fluids as plasma of many ruminants, but not of nonruminants, pregnancy plasma, seminal plasma, and amniotic fluid (Tabor, 1951; Hirsch, 1953b; Tabor et al., 1954; Blaschko and Hawes, 1959; Blaschko et al., 1959; Kobayashi, 1964; Caldarera et al., 1965, 1969; Yamada et al., 1967; Tryding and Willert, 1968; Argento-Cerh et al., 1973a,b; Beaven and De Jong, 1973; Janne et al., 1973; Bardsley et al., 1974; Nakano et al., 1974; Baylin and Margolis, 1975; Holtfa et al., 1975; Yasunobu et al., 1976; Holtta, 1977; A. C. Andersson et al., 1978a, 1980b; Illei and Morgan, 1979a,b, 1980; Bieganski et al., 1980; Gahl et al., 1980; Morgan, 1980; Morgan et al., 1980). Essentially, those enzymes capable of oxidizing or cleaving spermidine and spermine are collectively called polyamine oxidases, irrespective of whether they also act on mono- or diamines (Morgan, 1980). Only seldom is a monoamine oxidase able to attack polyamines. Additionally, a fraction purified from an L-aminoacid oxidase of viper venom has been recently demonstrated to oxidize polyamines as well (Braganca et al., 1979). However, it is not yet well established whether there is one enzyme capable of oxidizing all the polyamines or a separate enzyme oxidizing each. Some findings have supported the existence of separate specific

POLYAMINES IN MAMMALIAN TUMORS

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oxidases, from different sources, for each polyamine, i.e., a putrescine oxidase composed of two different subunits (Gahl and Pitot, 1979; M. Okada et al., 1979a,c; Gahl et al., 1980), a spermidine oxidase (M. Okadaet al., 1979b; Gahl et al., 1980), and a spermidine and spermine oxidase, collectively named polyamineoxidase (PAO) (Holtta, 1977). These reports are in keeping with earlier ones (Hirsch, 195313; Blaschko and Hawes, 1959; Blaschko et al., 1959; Bachrach, 1962). The topic of amine oxidases and the polyamine oxidases has been reviewed (Blaschko, 1962, 1963; Zeller, 1963; Buffoni, 1966, 1980; A. C. Andersson et al., 1980b; Morgan, 1980). The products of oxidative cleavage of the polyamines differ noticeably in relation to the source from which the enzyme employed was isolated (e.g., from mammalian or bacterial systems). 1. Those mammalian amine oxidases capable of oxidizing polyamines, (noticeably active in the intestines, placenta, ovary, semen, serum, and kidneys of various animals) catalyze the oxidative deamination of one or both terminal -CH2NH2 groups of the polyamines to the corresponding mono- or dialdehydes (Unemoto, 1963; Taboret al., 1964a,b). The products of the enzymic reaction are the following: an iminomonoaldehyde from the oxidation of spermidine or an iminodialdehyde from spermine, plus two molecules of ammonia and hydrogen peroxide in the case of spermine, and one molecule of ammonia and hydrogen peroxide in the case of spermidine (Unemoto, 1963; Tabor et al., 1964a). The dioxidation of spermine and the monooxidation of spermidine are represented by the following equations : H2N(CH2)sNH(CH2)iNH(CH2)3NH2+ 202 + 2H2O + Spermine 0 0

\\

H

/

//

+ 2NH3 + 2H20

C(CH2)2NH(CHp)dNH(CHz)&

'H Dioxidized Spermine

0-

HIN(CH~)$JH(CH&NH~ + 02 + HpO +

\\ /

C(CH~)~NH(CH~)INHI+ NH3 + HzO2

H' Spermidine

Monooxidized Spermidine

The products of oxidized polyamines were found to be quite unstable and to undergo successive and spontaneous metabolic cleavage because dioxidized spermine yields monooxidized spermidine and acrolein

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GIUSEPPE SCALABFUNO AND MARIA E. FERIOLI

CH,,=CH--C

// \

0 H

whereas monooxidized spermidine decomposes to putrescine and acrolein (Tabor et al., 1964a; Kimes and Morris, 1971a). The monoaldehyde derived from oxidation of spermidine can be metabolized to putreanine, a condensation product of putrescine and P-alanine, [H2N(CH2)4NH(CH2)2COOH, i.e., N-(4-aminobutyl)-3-aminopropionic acid], by an aldehyde dehydrogenase (Nakajima, 1973). Putreanine has been found in the brain and liver of mammals (Kakimoto et al., 1969; Shiba and Kaneko, 1969; Nakajima, 1973; Kremzner and Sturman, 1979). 2. Those bacterial amine oxidases capable of cleaving polyamines catabolize polyamines by splitting the molecules into smaller fragments. The different kinds of oxidative degradation of spermidine and spermine by bacterial suspensions entail the formation either of 1,3-diaminopropane and y -aminobutyraldehyde [H2N(CH2),CHO] (which spontaneously cyclizes to yield A'-pyrroline)

A'-Pyrroline

or of 1,4-diaminobutane and P-aminopropionaldehyde [or 3-aminopropionaldehyde, H2N(CH2)&H0, which is further oxidized to palanine, H2N(CH2)2COOH or converted to a dialdehyde, i.e., malondialdehyde, OHCCH2CHO]. This difference in the products of the catabolic reaction depends on the type of bacteria employed (Razin et al., 1958, 1959; Weaver and Herbst, 1958a,b; Bachrach et al., 1960; Bachrach, 1962; Padmanabhan and Kim, 1965; Okada et al., 1979b). An alternative origin of p-aminopropionaldehyde from 1,3diaminopropane has also been clearly demonstrated (Quash and Taylor, 1970). It is also worthwhile to note that some bacterial oxidases, mainly spermidine oxidase, do not utilize molecular oxygen but require the addition of electron acceptors; they are therefore called spermidine dehydrogenases rather than oxidases (Tabor and Kellogg, 1970; M, Okada et al., 1979b). 3. Polyamine oxidase (PAO) is a single flavin enzyme, newly identified from rat liver (Holtfa, 1977), which catalyzes the oxidation of both spermidine and spermine. These molecules are cleaved at the secondary amino groups to yield 3-aminopropionaldehyde and putrescine from spermidine or spermidine from spermine. Recently, it has

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also been shown that some acetyl derivatives of spermine and spermidine (namely,N ' - andN8-acetylspermidine,N1-acetylspermine, and N1,N1*-diacetylspermine)are natural substrates of this enzyme (Holtta, 1977; Seiler et al., 1980b). Undoubtedly, the mono- and dialdehydes, which arise from the extracellular enzymic oxidation of spermine and spermidine, are, from the biological point of view, more important than other catabolic derivatives of polyamines. In fact, dioxidized spermine has been shown to inhibit growth of various bacteria (Hirsch and Dubos, 1952; Taylor and Morgan, 1952; Hirsch, 1953a; Tabor et al., 1954; Tabor and Rosenthal, 1956; Razin and Rozansky, 1957; Bachrach and Persky, 1964; Kimes and Morris, 1971b),to have a phagocidal action (Bachrachet aZ., 1963, 1971; Bachrach and Leibovici, 1965a,b, 1966; Fukami et al., 1967; Oki et aZ., 1968, 1969), and to inactivate animal and plant viruses (Bachrachet al., 1965,1971; Bachrach and Don, 1970,1971; Kremzner and Harter, 1970; Bachrach and Rosenkovitch, 1972). It is important to recall here that it has been realized since the earliest investigations (Hirsch, 1953a) of this topic that the presence of an appropriate plasma or seminal liquid in the incubation medium was essential for rendering spermine inhibitory for the bacterial growth, since spermine did not inhibit the multiplication of tubercle bacilli in a synthetic medium without plasma. In this regard, it is likely that the oxidized polyamines can be part of the mechanisms of native aspecific immunity, as factors present in blood and in tissues that are active against bacterial and viral growth. Additionally, the enzymatically oxidized polyamines cause a loss of motility of sperm cells and powerfully inhibit some important metabolic processes in the spermatozoa (Tabor and Rosenthal, 1956; Janne et al., 1973; Pulkkinen et al., 1978). Finally, the oxidation products of spermine and spermidine are powerful negative effectors of cellular proliferation, as has been demonstrated in cultures of fibroblasts (Gahl et al., 1976; Gahl and Pitot, 1978; Gaugas and Dewey, 1979; Webber and Chaproniere-Rickenberg, 1980), mitogen-stimulated lymphocytes (Byrd and Jacobs, 1977; Byrd et al., 1977, 1978; Gaugas and Curzen, 1978; Allen et al., 1979; Gaugas and Dewey, 1979; Gaugas, 1980b; Swanson and Gibbs, 1980), and unstimulated thymocytes (Gaugas and Dewey, 1979; Morgan et al., 1980). The interaction of polyamine oxidase with one of the new bacterial polyamines, i.e., thermine, has also been shown to potently inhibit lymphocyte proliferation (Gaugas and Dewey, 1979; Gaugas, 1980b). The activity of these drugs against bacteria and viruses can be explained by their binding to cellular nucleic acids and by their capacity

190

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to inhibit the syntheses of nucleic acids and mRNA in microorganisms and in mammalian cells (Bachrach and Persky, 1966, 1969; Bachrach and Eilon, 1967, 1969a,b; Persky et al., 1967; Eilon and Bachrach, 1969).As was cleverly ,suggested by Bachrach and his co-workers, who excellently and thoroughly investigated the products of oxidative catabolism of polyamines and their biological properties, these monoand dialdehydes resemble bifunctional alkylating agents, such as nitrogen mustards, and also behave like those antibiotics that inhibit DNA-directed RNA polymerase by binding to the DNA template. The biological actions of short-chain aliphatic aldehydes on different eukaryotic and prokaryotic systems have been extensively studied in our institute by Ciaranfi and his co-workers, who have obtained some important results (such as the inhibition of protein synthesis and of cellular proliferation, especially in neoplastic cells, and the essential nature of the presence of an aldehydic group for the inhibitory properties of the molecules), and these actions are analogous to those of the long-chain mono- and dialdehydes arising from oxidation of spermidine and spermine (Guidotti et al., 1964,1965; Ciaranfi et al., 1965, 1971; Perin et al., 1972, 1978; Sessa et al., 1977). Thiazolidin-4carboxylic acid, which is the condensation product of some aliphatic aldehydes with L-cysteine both in vitro and in vivo (Guidotti e t al., 1965; Loreti et al., 1971), is one of the pharmacological agents which has recently been found to be able to restore “contact inhibition” in tumor cell cultures (Gosalvez et al., 1979). Some very exciting perspectives about the biological actions and significance of the oxidized derivatives from polyamines have been put forward by some investigators. On the grounds of the experimental data showing high and increasing activity of DAO in the placenta and plasma of mammals during pregnancy (Kobayashi, 1964; Tryding and Willert, 1968; Bardsley et al., 1974; Baylin and Margolis, 1975; A. C . Anderson et al., 1978a; Illei and Morgan, 1979a,b), a high polyamine content of mammalian placenta (Porta et al., 1978), and, finally, the powerful suppression of the mitogen-stimulated lymphocyte proliferation elicited in vitro by the products of the ruminant sera-polyamine interaction, it has been argued that such an immunodepressant effect might also operate in vivo in the intervillous circulation of the placenta. Such a mechanism might represent a natural and localized immunological barrier, which can protect, at least in part, the conceptus from immunological rejection by the mother caused by the immunological incompatibility between the fetoplacental unit and the mother (Byrd et al., 1978; Gaugas and Curzen, 1978; Morgan and Illei, 1980). Further credence is given to such a hypothesis by the following

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findings: (1)high levels of ODC activity were found in the developing rat placenta; (2) most of the ODC activity was found in the fetal part of the rat placenta; (3)in the rat placenta during pregnancy, the time course of ODC activity was similar to that of DAO activity; (4) in contrast to the pattern of distribution for ODC activity, most DAO activity was found to be localized in the maternal part of the rat placenta (Maudsley and Kobayashi, 1977). Although P-aminopropionaldehyde has been identified as a normal constituent of human serum (Quash and Taylor, 1970) and acrolein has been demonstrated to be formed in significant amounts from enzymatically oxidized spermine and spermidine in vitro (Alarcon, 1966,1970, 1972),the physiological significance of the former aldehyde remains to be established, whereas the cytotoxic property (Alarcon, 1966, 1970, 1972) and the antimicrobial activity (Kimes and Morris, 1971b) of the latter aldehyde have been clearly demonstrated, although its antiviral activity is still highly doubtful (Bachrach et al., 1971; Bachrach and Rosenkovitch, 1972). Furthermore, the real role of acrolein as possible inhibitor of cell proliferation has also been questioned (Gaugas and Dewey, 1979; Gaugas, 1980b). Finally, a further possible elucidation of the molecular mechanism involved in the cytostatic effect of the PAO-spermine interaction has been proposed by Gaugas and Dewey (1980).Accordingly, 02-.produced during oxidative catabolism of spermine by PA0 could react with dioxidized spermine to generate free radicals that could be additional antimitotic agents. The production of 02-dependent free radicals or H202, also generated by polyamine catabolism, is apparently not directly involved in in vitro cytostatic effect, since this effect was found to be independent of catalase activity and the addition of superoxide dismutase paradoxically potentiated that effect. The topic of oxidized spermidine and spermine and their biological roles has been thoroughly reviewed several times by Bachrach (1970a,b, 1973). 2. Oxidative and Nonoxidative Catabolism of Putrescine and Cadaverine In mammals, putrescine catabolism occurs through several multistep pathways. 1. First, the oxidative deamination of putrescine involving mammalian DAOs leads to y-aminobutyraldehyde (4-aminobutyraldehyde) formation, which nonenzymically cyclizes into an internal aldimine ring, A1-pyrroline. One molecule each of ammonia and hydrogen peroxide are also formed during this reaction. This direct pathway

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

catalyzed by DAO is quantitatively the most important. Putrescine oxidase from some bacteria also cleaves putrescine to give the same The catabolic oxidative pathway of final products (Okadaet al., 1979~). putrescine is presented in the following scheme: HzN(CH2)dNHZ + HzO + Putrescine

+ HzOp + NHD y-Aminobutyraldehyde

0 2 + HzN(CH&CHO

A'-Pyrroline

However, from a theoretical point of view, y-aminobutyraldehyde will only be quantitatively transformed into A'-pyrroline when the tissue which contains the DAO activity is utterly devoid of any aldehyde-metabolizing enzymes. Most mammalian tissues are able to metabolize y-aminobutyraldehyde as substrate. This aldehyde can be oxidized by an aldehyde dehydrogenase to yield y-aminobutyric acid (GABA) or reduced by an aldehyde reductase or an alcohol dehydrogenase to yield 4-amino-l-butanol (Fogel et al., 1978). In recent years, an ever-increasing number of reports on the conversion of putrescine to GABA in vivo and i n vitro in different organs of mammals have appeared (Seiler and Knodgen, '1971; Seiler et al., 1971, 1973a, 1979a; Seiler, 1973; DeMello et al., 1976; Henningsson and Rosengren, 1976; Konishi et al., 1977; Tsuji and Nakajima, 1978; Sobue and Nakajima, 1978; Anderson and Henningsson, 1980a; Andersson et al., 1980a). It has also been shown in these reports that the transformation of putrescine into GABA implies the presence of a direct pathway which does not include glutamic acid as an intermediate, since it is well established that in eucaryotes GABA can also be synthesized from glutamic acid, catalyzed by L-glutamate l-carboxy-lyase (or glutamic acid decarboxylase, EC 4.1.1.15). The enzyme mainly involved in the in vivo conversion of putrescine to GABA is again DAO (Seiler and Eichentopf, 1975; Sourkes and Missala, 1978; Tsuj and Nakajima, 1978). Evidence has been presented that ornithine is also a precursor for GABA in adult mammalian tissues, probably via putrescine (Seiler and Knodgen, 1971; Murrin, 1980). Recently, the regulatory interrelations between polyamines and GABA have been carefully reconsidered by Seiler et al. (1979b, 1980a). 2. Experimental evidence has been accumulated in favor of the existence of another catabolic pathway for putrescine, in which this diamine is first acetylated to monoacetylputrescine, which is, in turn, degraded to GABA, through the different steps shown in Fig. 5 (Seiler

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POLYAMINES I N MAMMALIAN TUMORS

N -acetylS-AB-CHO

1

ALD O H

ALD-DH

0 R

0

SI I

N - a t e l yI S-AB-COOH

a enzyme

COOH

r-AB-COOH I I

b Succinic acid

C t t r a t e cycle

COt

1 , 4 - 0 A B = 1.4-DIAMINOBUTANE (PUTRESCINE ) r - A B - C O O H = r-AMINOBUTYRIC ACID r - A B - C H O = r-AMINOBUTYRALDEHYDE M A 0 AMlNE OXIDASE ( F L A V I N - CONTAINING) ( E C 1 4 3 4 ) OAO = AMlNE O X I D A S E (COPPER-CONTAINING) ( E C 1 4 3 6 ) A L O - O H = AMINOBUTYRALDEHYDE DEHYDROGENASE (EC 1 2 1 1 9 ) ACETYLATING ENZYME = PUTRESCINE ACETYLTRANSFERASE (EC 2 3 1 57) DEACETYLATING ENZYME = N-ACETYL 4-AMINOBUTYRIC AClO DEACETYLASE

@ @

EXTRAMITOCHONDRIAL PATHWAY MITOCHONDRIAL PATHWAY

FIG.5. Different pathways of putrescine catabolism in mammalian tissues.

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GIUSEPPE SCALABRINO AND MARIA E. FEFUOLI

and Al-Therib, 1974a,b; Seiler and Eichentopf, 1975; Seiler et al., 1979a). The enzyme acetylating putrescine, i.e., the acetyl-CoAdependent N-acetyltransferase, was found to be located mostly in the nucleus (Seiler and Al-Therib, 1974a; Seiler et al., 1975). However, this catabolic pathway of putrescine is probably a minor one. In summary, one can state that two of the three main catabolic routes for 1,Cdiaminobutane in mammalian cells lead to the formation of GABA, which in the extramitochondrial compartment, is practically a metabolic end product but which in the mitochondrial compartment is an intermediate product, further metabolized in the citrate cycle. Surprisingly enough, it should be noted that, at least in the brain, the mitochondrial catabolic pathway for putrescine includes a step in which a typical diamine is deaminated by MA0 (Seiler and Al-Therib, 1974b). This peculiar behavior of M A 0 has recently been confirmed for the acetyl derivative of another diamine, i.e., monoacetylcadaverine, deaminated by rat liver M A 0 (Suzuky et al., 1980). 3. A third catabolic pathway of putrescine has been detected and demonstrated to be independently active, though only in some mammalian organs. In this case, putrescine is converted via A'-pyrroline to 2-pyrrolidone (a lactam not previously identified in any biological system) in liver, spleen, and lung but not in kidneys, brain, heart, and muscle (Lundgren and Hankins, 1978; Lundgren et al., 1980). This lactam is then metabolized into 5-hydroxy-2-pyrrolidone7another compound new to the biological systems (Lundgren and Fales, 1980). Nevertheless, this newly identified catabolic route for putrescine needs deeper and more careful studies to clarify the enzymatic basis of the formation of these derivatives. The three main catabolic pathways of putrescine in mammals are presented in Fig. 5. 4. Putrescine may also be converted into y-aminobutyraldehyde by a nonoxidative mechanism, catalyzed by a diamino-a-ketoglutarate transaminase, present in a variety of bacteria (Kim and Tchen, 1962; Kim, 1964; Michaels and Kim, 1966). In this enzymic reaction, free ammonia is not released nor is oxygen consumed. 5 . Finally, 1,s-diaminopentane is oxidized by animal DAO to A'piperidine with production of ammonia and hydrogen peroxide and with oxygen consumption (Bachrach, 1973), according to the following t

0 2+

HeN(CH2)rCHO + HzOZ + NHS

6 N

Hzo

A'-Piperidine

POLYAMINES IN MAMMALIAN TUMORS

195

F. CONJUGATION PRODUCTS AND EXCRETION PRODUCTS As far as the conjugation products of polyamines are concerned, quite similar conjugated forms have been identified in prokaryotes and in eukaryotes. In fact, in bacteria, polyamines can be conjugated with glutathione or be acetylated (mono- or diacetylated) or linked to peptides (Dubin, 1959; Dubin and Rosenthal, 1960; C. W. Tabor and Tabor, 1970; H. Tabor and Tabor, 1975, 1976). This is also essentially true for the eukaryotes. Monoacetylputrescine [ HzN(CHz),NHC CHa](N’-acetyl-l,4-diaminobutane)

1I

0

has been found in vertebrate tissues (Seiler et al., 1973b), in human brain (Perry et al., 1967), in normal human lymphocytes (Menashe et al., 1980), and in the urines of normal humans and rats (Noto et al., 1978; Seiler and Knodgen, 1979b). The presence of such a molecule must be connected with that catabolic pathway of putrescine which begins with the acetylation of this molecule. A y-glutamyl derivative of putrescine has been detected in rat brain (Nakajima et al., 1976). Both spermidine and spermine, but not diamines, have been demonstrated to serve as substrates for acetyl-CoA-dependent N acetyltransferase activity present in the nuclei of liver of calf and rat and in isolated chromatin from rat liver and kidney, and undergo N-acetylation in this system (Blankenship and Walle, 1977; Libby, 1978a, 1980). In the case of spermidine, the product of the enzymatic reaction is acetylspermidine B (or N1-acetylspermidine). This compound can be further metabolized by PA0 (Holtta, 1977), whereas acetylspermidine A (or iV-acetylspermidine) is deacetylated by a specific cytosol enzyme present in rat tissues to yield acetic acid and spermidine (Blankenship, 1978; Blankenship and Walle, 1978). N 1 Acetylspermidine, alternatively, can be enzymatically converted to putrescine (Blankenship, 1979) or enzymatically deacetylated (Libby, 1978b).

!

H,N(CHz)aH(CHz)$IH CHS N ‘-Monoacetylspermidine [ N ‘-(3-acetamidopropyl)-1,4diaminobutane]

E

HSC NH(CHz)4NH(CHz)sNHp N8-Monoacety lspermidine [N-(4-acetamidobutyl)-1,3diaminopropaneJ

Monoacetylspermidines (A and B forms) are normal constituents of human urine (Nakajima et al., 1969; Noto et al., 1978; Seiler and

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

Knodgen, 1979b). l-N-Acetylspermidine occurs normally in human serum (Smith et al., 1978) and in human lymphocytes (Faber et al., 1980; Menashe et al., 1980). Putreanine and N-(3-aminopropyl)-4aminobutyric acid are other normal catabolites of spermidine in human urine (Noto et al., 1978; Asatoor, 1979). The occurrence of the diamines (putrescine and cadaverine) and polyamines (spermidine and spermine) in human urine as Schiff bases, in which the polyamines conjugate with pyridoxal or pyridoxal phosphate has recently been recognized (Aigner-Held et al., 1979). In the blood of normal subjects, cadaverine is present as monoacyl derivatives, i.e., monoacetylcadaverine and monopropionylcadaverine (Dolezalova et al., 1978). The products of conjugation of polyamines with peptides in some biological fluids, such as human plasma and human amniotic fluid, have been very carefully investigated by Rennert and his group. These investigators found that the polyamines are conjugated to peptide carriers (Chan et al., 1978, 1979; Seale et al,, 1979a,b). In plasma, this peptide conjugate contains, in largest amount, putrescine and a small amount of spermidine, but no spermine (Seale et al., 1979a). In the amniotic fluid, putrescine, spermine, and spermidine are associated with peptides, but spermine can also be present as the acetylated derivative (Chan et al., 1978,1979; Seale et al., 1979b). Putrescine can also be transported in human plasma bound to fibronectin (Roch et al., 1980). Conjugation is a process not involving any structural alteration of polyamines and is a biochemical reaction well known to occur typically in tbe liver in uiuo. On the one hand, no conjugation of polyamines of any kind takes place in vitro between labeled polyamines and plasma or whole blood (Rosenblum et al., 1976), and, on the other hand, near-total hepatectomy of the rat prevents the formation of detectable polyamine conjugates (Rosenblum et d., 1976; Rosenblum and Russell, 1977). Although acetylated derivatives of polyamines are probably the most significant fraction of the conjugated polyamines, the newly identified polyamine-peptide conjugates may provide important new insights into the various metabolic transformations of polyamines in man and other mammals, both healthy and diseased. Therefore, one can conclude that-as far as is known-the free polyamines are present in the physiological body fluids of mammals in small or even in negligible amounts, with the conjugated forms clearly quantitatively predominant (Janne et al., 1978). However, whether acetylated polyamines play active biochemical roles, are only physio-

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logically inactivated polyamines, or are only excretion forms of these compounds [it is noteworthy that polyamine acetyltransferase has also been found in kidney (Blankenship and Walle, 1977)] remains to be worked out. Polyamines are also included in the molecular structures of some antibiotics and some antineoplastic agents. Edeine A contains spermidine in covalent linkage to a pentapeptide (Hettinger and Craig, 1968; Hettinger et al., 1970). Edeine B contains N-guanyl-N’-(S aminopropyl)-1,4-diaminobutane (guanylspermidine) (Hettinger et al., 1968, 1970; Kid0 et al., 1980). Among the active bleomycins, which are a group of natural and synthetic antitumor drugs, bleomycin A5 contains the triamine spermidine and bleomycin & the tetraamine spermine (Cohen and I, 1976; Lapi and Cohen, 1977; Cohen, 1979; Kid0 et al., 1980). G. NATURALANTIPOLYAMINEANTIBODIES Normal human and rabbit sera have been shown to contain naturally occurring antipolyamine antibodies. These natural antibodies react specifically with polyamines and have been classified as IgG immunoglobulins, regardless of the species in which they have been identified (Roch et al., 1978,1979; D. Bartos et aZ., 1980; Ripoll et al., 1980). The biological significance of these antibodies and their susceptibility to quantitative change in different kinds of diseases, including neoplasms, will be of theoretical and practical concern. II. Levels of Polyamines and Their Biosynthetic Enzymes in Fully Developed Experimental Tumors

The aim of this section is to summarize present knowledge about the relationships between polyamines and some fully developed experimental tumors. For this purpose, we will consider the changes in polyamine content observed in different types of tumor, the alterations in polyamine biosynthetic decarboxylase activities, and the applicability of these parameters as biological markers of tumor growth or regression. Starting from the earliest reports (Russell and Snyder, 1968; Snyder and Russell, 1970; Snyder et al., 1970), which showed high levels of ODC activity in some hepatomas and sarcomas, a great deal of research has been done in this specific area. It is necessary to state as a preliminary that, in analyzing the polyamine contents of neoplasms, one needs to establish their physiological levels and the normal changes in those levels in the corresponding normal tissues before one can correlate increases of these

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

substances with neoplastic transformation or with different degrees of malignancy. Ornithine is an interesting amino acid since it appears not to be a true building block in the biosynthesis of any major protein, but it is a key metabolite for which several enzyme systems and metabolic pathways compete. In the livers of mammals, ornithine is produced by the action of arginase on L-arginine and is thereafter utilized in several metabolic pathways: it may be (1)channeled by ODC into polyamine synthesis, (2) siphoned off into the urea cycle by ornithine transcarbamylase (OCT), and (3) metabolized by the transaminase reaction catalyzed by L-ornithine : 2-oxoacid aminotransferase (ornithine transaminase) (OTA) into glutamic-y-semialdehyde synthesis. Of the enzymes involved in the metabolic pathways of ornithine, only ODC is at present known to be constantly increased in rapidly growing rat liver tumors, and its increase has been positively correlated with the growth rate of hepatomas tested. In fact, a systematic study carried out with several rat hepatomas with different growth rates resulted in the discovery of a shift in ornithine metabolism that could confer a selective biological advantage on the cancer cell and that is linked with tumor growth rate (Weber, 1972, 1973a,b; Weber et al., 1972a,b; Williams-Ashmann et al., 1972a,b, 1973; Tomino e t al., 1974). The term “imbalance” was applied to this shift by Weber to indicate alterations from the normal state of metabolic balance, which denotes dynamic equilibrium in steady-state conditions. For the metabolic pathways of ornithine in rat hepatomas, the imbalance is represented by progressively elevated ODC activity, in parallel with the increase in the hepatoma growth rate, whereas OTA and OCT activities progressively declined in parallel with the increase in the hepatoma growth rate. This imbalance in ornithine metabolism in tumor cells was accompanied by other imbalances in the metabolic pathways of carbohydrates, pyrimidine, and cyclic nucleotides (Weber, 1972, 1973a,b; Weber et al., 1972b). Consequently, as a result of these quantitative modifications of the activities of the three key enzymes (ODC, OTA, OCT) which compete for ornithine, in the rapidly growing neoplastic hepatomas only the one (ODC) that channels ornithine into the polyamine biosynthetic pathway is selectively increased, and the other two (OTA, OCT) are selectively and markedly decreased. Therefore, the biochemical advantages that arise from the imbalance in ornithine metabolism and are able to support the high growth rate of the hepatoma cells are essentially two, occurring concomitantly: (a) increased polyamine biosynthesis, starting with the increased ODC activity; (b) increased purine and pyrimidine biosynthesis, due to the

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decreased OCT activity, which decrease allows lesser utilization of carbamyl phosphate and aspartate in the urea cycle and increased utilization for the synthesis of DNA and RNA. It is well known, in fact, that the urea cycle competes with purine and pyrimidine biosynthesis for two important precursors, carbamyl phosphate and aspartate. However, it is still an open question whether such a derangement of the activities of the three enzymes that compete for ornithine utilization is typical of all neoplasms or is characteristic only of the hepatomas, since no other such systematic studies are available for neoplasms of other organs. However, the simultaneous presence of all these imbalances in carbohydrate, pyrimidine, nucleic acid, ornithine, and cyclic nucleotide metabolism appears to be specific for neoplastic liver, since no similar patterns have been found in fetal, normal, differentiating, or regenerating rat liver (Weber, 1972, 1973a,b; Weber et al., 197213). Two other pertinent considerations must be added: (1)the SAMD activities of almost all the hepatomas tested were either within the normal range or even decreased, only very rarely being higher than in normal liver; (2) putrescine concentrations generally tended to be highest in the most rapidly growing hepatomas, but the steady-state concentration of this diamine did not always exactly mirror the ODC activity of the same sample of tumor tissue (Williams-Ashman et al., 1972a,b, 1973). Furthermore, in some Morris hepatornas, ODC levels were also found to be dissociated from concentrations of spermidine and spermine, which were even lower than those in resting liver (Cavia and Webb, 197213). These findings are particularly important because these reports are among the earliest to stress the possibility that ODC, rather than the polyamine content or pattern, might be a better indicator of the growth rate of a neoplastic tissue. However, Pariza et al. (1976a) questioned whether the increase in ODC activity is a good indicator of the hepatoma growth rate, because they found in circadian rhythm studies that ODC activity in Morris hepatoma 7800 was lower than in normal liver throughout most of the day. For the same hepatoma, Cavia and Webb (1972a,b) reported just the opposite, with the ODC activity level higher than in control liver. The ODC level of the same hepatoma was decreased after bilateral adrenalectomy of the host, demonstrating the ODC, at least in this case, was still hormone responsive (Cavia and Webb, 1972a). The picture is further complicated by the fact that another indicator for the growth rate of the neoplasms has been proposed, the spermidine/spermine ratio (Russell, 1973b; Russell and Durie, 1978). Elevation of this biochemical ratio, whether or not accompanied by high putrescine content, has been demonstrated to be greater in fast-

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GIUSEPPE SCALABRINO AND MARIA E. FERTOLI

growing hepatomas than in slow-growing ones (Russell, 1973b; Russell and Durie, 1978) and to occur also in both solid and ascites Rd13 sarcoma (Neish and Key, 1967, 1968), in Ehrlich ascites tumor cells, in rat mammary tumor, and in L1210 leukemia (see discussion later in this section). It is noteworthy that spermine stimulated in uitro the activities of all three RNA polymerases from a rapidly growing hepatoma when native DNA was used as template and, even more, that the stimulation of all these tumor enzymic activities was greater than those of the corresponding activities from normal rat liver (Rose et al., 1976). The stimulation by spermine of DNA-dependent RNA-polymerase activity has been confirmed in in uitro studies with enzyme purified from nuclei of Ehrlich ascites tumor cells (Blair and Mukherjee, 1973). Some other effects induced by polyamines at the molecular level have been investigated in some Morris hepatomas. Spermine stimulated the total ADP ribosylation of nuclear proteins in two Morris hepatomas and in host liver, while the effect of spermidine was less marked (Perrella and Lea, 1979). Moreover, the effect of spermine on the relative proportion of labeled ADP-ribose incorporated into the different nuclear fractions was to further strengthen the trend, already evident in its absence, in a spectrum of homogeneous tissues with different proliferation rates (such as resting liver, regenerating liver, and some Morris hepatomas), to increase the incorporation of ADPribose into nonhistone proteins, and to decrease concomitantly the incorporation into histones (Perrella and Lea, 1979). Further studies focused on the effects of spermine on the distribution of ADP-ribose molecules bound to histones in hepatomas and host liver. Interestingly, spermine once again caused a shift in the incorporation of labeled ADP-ribose in the histones of the nuclei from hepatomas and host liver (Perrella and Lea, 1979). In detail, spermine caused a shift in the distribution of labeled ADP-ribose from the core histones (i.e., the histones which form the core of the nucleosomal structure) to the H 1 internucleosomal histones, without significantly changing total incorporation into histones (Perrella and Lea, 1979). This is another indication of the role of spermine in regulating DNA-to-protein interactions. Ehrlich ascites carcinoma, growing in the peritoneal cavities of mice, is a tumor particularly well studied for polyamine biosynthesis and content. The putrescine concentration in the tumor cells markedly increased in the first week after tumor inoculation, i.e., during the phase of extremely rapid cell multiplication, but thereafter it dropped by nearly one-half (Anderson and Heby, 1972, 1977). This time course

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was not confirmed by Kallio et al. (1977d), who observed only small changes in cellular putrescine concentrations during tumor growth. The patterns of change in the intracellular concentrations of spermidine, spermine, DNA, and RNA were quite well correlated reciprocally with each other (Andersson and Heby, 1972). The spermidine concentration was found by some authors (Bachrach et al., 1967; Andersson and Heby, 1972; Kallio et al., 1977d; Uehara et al., 1980), but not by others (Siimes and Janne, 1967), to be always much higher than that of spermine. These data lend further support to possible connections of putrescine concentration with rapid growth phase and of spermidine and spermine concentrations with nucleic acids. Indeed, a high positive correlation between the cellular content of spermidine and spermine and that of nucleic acids has been reported (Andersson and Heby, 1977). In this respect, spermidine stimulated in vitro the RNA synthesis of Ehrlich ascites tumor cells when it was present in the incubation medium at low concentrations, but it had exactly the opposite effect at higher concentrations (Raina et al., 1968). The ability of Ehrlich ascites cells to form spermidine from putrescine in uivo and to interconvert spermidine and spermine in ui tro was also demonstrated in early studies of this type of tumor (Bachrach et al., 1967; Siimes and Janne, 1967). As for the activities of the polyamine biosynthetic enzymes in Ehrlich ascites cells during tumor growth, ODC and SAMD were found to change very little during the first week after the i.p. inoculation, whereas spermidine and spermine synthases increased significantly, in the last 3 days of the same period (Kallio et al., 1977d). In contrast with this, ODC activity is markedly increased in Ehrlich ascites tumor cells, reaching a peak 4 days after tumor inoculation, i.e., at nearly the time when cell proliferation is fastest (Noguchi et al., 1976a,b). More careful sequential studies demonstrated that ODC activity increased dramatically within a few hours after the i.p. inoculation of the ascites tumor cells (Harris et al., 1975; Andersson and Heby, 1977; G. Andersson et al., 1978b). It was also possible to establish that there are high positive correlations between the activities of ODC and SAMD and the specific growth rate of the tumorous cells (Andersson and Heby, 1977, 1980; G. Andersson et al., 1978b) and between the peak in ODC activity and the peak of the [3H]thymidine index, i.e., the ODC increase paralleled the surge of cells from the Gl-Go into the S phase (Harris et al., 1975). Very surprisingly, when the activities of the two polyamine biosynthetic decarboxylases began to decline, the decrease was not accompanied by a concomitant decrease in the cellu-

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lar spermidine and spermine contents, which instead continued to rise during plateau phase growth, when the tumor growth rate had decreased (G. Andersson et al., 1978b; Andersson and Heby, 1980). From E hrlich ascites tumor cell-free fluid, a factor has been purified that is a heat-labile, alkali-stable, acidic protein and is able to stimulate in uiuo ODC activity in liver and spleen but not in kidneys of normal mice (Noguchi et al., 1976a,b; Kashiwagi et al., 1979). ODC induction by this factor had a very slow time course after in uiuo injection and seemed to occur independently of a release of hypophyseal or adrenal hormones, since induction was also observed in hypophysectomized or adrenalectomized mice (Noguchi et al., 1976a; Kashiwagi et al., 1979). The ODC-stimulating factor appears to be specific for Ehrlich ascites tumor fluid and cells, since in vivo injection of homogenates of various normal tissues into mice caused no stimulation of ODC activity in the livers of the recipient normal animals (Noguchi et al., 1976a). This factor, released from tumor cells, is also thought to be responsible for the observed increases in ODC activity in the liver and spleen of the host animal a few days after the i.p. inoculation of Ehrlich ascites tumor cells, since these changes were not accompanied by infiltration or metastasis of tumor cells in these organs (Noguchi et al., 1976a,b). In contrast, the renal ODC activity in mice with Ehrlich ascites carcinoma greatly decreased during the tumor growth in the days after the inoculation (Noguchi et al., 1976a,b). According to an attractive hypothesis (Andersson and Heby, 1977, 1980; Heby et al., 1979; Linden et al., 1980), the extracellular polyamines released into the ascites fluid as a result of tumor cell death could be responsible for the inhibition of intracellular polyamine biosynthesis, which in turn brings about a marked reduction in the rate of DNA synthesis and cell proliferation (see also Section 111,E). Putrescine, when administered in uiuo in repeated injections, strongly inhibited the tumor cell proliferation and ODC activity by eliciting the formation of the ODC antizyme, and, when added to the in vitro assay mixture, it markedly inhibited ODC activity in the cytosol fraction from tumor cells stimulated to grow (Andersson and Heby, 1977,1980; Hebyet al., 1979; Lindenet al., 1980). On the other hand, multiple injections of putrescine into mice with Ehrlich ascites carcinoma cells neither affected the SAMD activity of neoplastic cells nor decreased their incorporation of tritiated thymidine into DNA (Linden et al., 1980). A factor not yet chemically identified and not tumor specific, but able to stimulate ODC activity in uitro has been demonstrated in ascites fluids taken from murine hosts at progressive stages of growth

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of different types of tumor, both chemically induced and spontaneously generated, e.g., fibrosarcoma, mammary carcinoma, ovarian carcinoma, leukosis L1210 (Vaage and Agarwal, 1979). This ODC stimulation was observed in the same neoplastic cells first isolated from the foregoing neoplasms grown in uivo and cultured in uitro. This stimulation of ODC by ascites fluid was greater during the initial period of malignant growth and then declined as growth continued, the only exception being leukemia L1210 (Vaage and Aganval, 1979). In serum and in ascites fluid from mice with malignant tumors, namely, fibrosarcoma, mammary carcinoma, and ovarian carcinoma, a “negative” factor with a markedly depressive effect on ODC activity of both normal unstimulated lymphocytes and PHA-activated lymphocytes is present (Vaage et aZ., 1978). An analogous inhibitory effect on ODC activity of PHA-activated lymphocytes taken from tumor hosts was also displayed by ascites fluids taken from mice given neoplastic implants (Vaage et al., 1978). This ODC inhibition was more pronounced, the more advanced the neoplastic disease (Vaage et d., 1978). Whether such factor(s) in serum and/or ascites fluid taken from tumor hosts could explain the general anergy frequently associated with advanced cancer remains a matter of speculation. In addition to rat hepatomas and Ehrlich ascites tumor cells, rat mammary carcinoma and mouse leukosis are ideal for studies of polyamine biosynthesis and accumulation. The ratio of spermidine to spermine in rat mammary carcinoma was higher when the tumor was growing (Russell et al., 1974c; Russell and Durie, 1978). Polyamine concentrations in rat mammary carcinoma were profoundly modified by castration plus removal of the pituitary gland, which induced the process of tumor regression. Putrescine and spermidine levels dropped significantly shortly after surgery, whereas the spermine level, surprisingly, increased (Russell et al., 1974c; Russell and Durie, 1978). Consequently, the spermidine/spermine ratio fell. In the tumor’s interstitial fluid, an increase in spermidine level was detected during tumor regression, whereas putrescine concentration was essentially unchanged (Russell et al., 1974c; Russell and Durie, 1978). In L1210 leukemia, both solid and ascites forms, ODC activity increased rapidly within a few days after inoculation and then dropped to very low levels just prior to the death of the mice (Russell, 1970; Russell and Levy, 1971; Russell and Durie 1978). SAMD activity was also elevated in the earliest phases of tumor growth, but, unlike ODC, it remained at high levels (Russell, 1970; Russell and Levy, 1971; Russell and Durie, 1978). As for polyamine concentrations, a high spermidine/spermine ratio in L1210 leukemia was due to an increase

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in the concentration of the former, concurrent with a slight decrease in the latter (Russell and Levy, 1971; Newton et al., 1976; Russell and Durie, 1978). The activities of nuclear DNA-dependent RNA polymerases I, 11, and I11 isolated from L1210 leukemia cells were stimulated by spermine more than by spermidine (Blair et al., 1980). In AKR leukemic cells, the spermidine and putrescine contents were similar in the Goand G1phases of the cell cycle, whereas the spermine level decreased from Go to GI (Heby et al., 1973). Furthermore, the cellular contents of three main polyamines increased progressively as the cell traversed the cell cycle phases from Gl to M (Heby et al., 1973). Ornithine metabolism has been measured in control animals and in rats with either Walker 256 carcinoma or methylcholanthrene-induced tumors by measuring the labeled COz liberated from [ l-14C]ornithine injected i.p. (Buftkin et al., 1978).This follow-up study revealed that ornithine metabolism was significantly increased over that in normal animals and that this increase can reasonably be connected with the growth rate of the neoplasm since the regression of the tumor occurred concurrently with a reduction in ornithine metabolism (BufIkin et al., 1978). Last, but not least, it is very interesting that some isozymes of S-adenosyl-L-methionine synthetase have been identified in some hepatomas as well as during chemical hepatocarcinogenesis and that no isozymes of the enzymes involved in the polyamine biosynthetic pathway have been identified so far in neoplasms. Briefly, there are two widely accepted classifications of the isozymes of Sadenosyl-L-methionine synthetase: (1)they are distinguished by their molecular weights and sensitivities to dimethylsulfoxide into a,p, and y forms; (2) they are distinguished by their different K, values for methionine and are called low K, intermediate K,, and high K, forms. The activities of the a-and /3-isozymes progressively decreased, whereas the kidney-type y-isozyme (which is present in kidney, in most other tissues, and in fetal liver) progressively increased during the rat liver carcinogenesis by N-2-fluorenylacetamide (Tsukada and Okada, 1980), in some Morris rat hepatomas, and in Yoshida ascites hepatoma AH130 (G. Okada et al., 1979). A neoplastic abnormal isozyme of S-adenosylmethionine synthetase, with an intermediate K,, has been found in neoplastic nodules induced by a hepatocarcinogen and in various transplantable hepatocellular carcinomas (Liau et al., 1979a,b). This type of isozyme has been detected in a large spectrum of human malignant tumors xenografted into athymic nude mice, but it has never been detected in any normal human tissue examined (Liau et al., 1980).

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Ill. Modification in Vivo and in Vitro of Tissue Polyamine Metabolism by Chemical Carcinogens and Tumor Promoters

A. EFFECTSOF A SINGLE ADMINISTRATIONOF A CARCINOGEN OR TUMORPROMOTER ON POLYAMINE BIOSYNTHESIS AND CONTENT IN THE TARGETTISSUES Generally speaking, a single administration of a carcinogenic drug (whether or not at carcinogenic dosages) or of a tumor promoter makes it easier to determine the molecular events occurring in the cells after penetration ,of the drug. Among hepatocarcinogenic molecules, a single injection of thioacetamide, a weak carcinogenic agent that stimulates RNA synthesis, is able to induce in vivo a large increase in hepatic ODC activity in the rat (Fausto, 1969,1970; Raina and Janne, 1970b; Cavia and Webb, 1972a; Ono et al., 1972,1973; Pegg et al., 1978). This kind of induction presents some particular features: (1)its peak occurs much later than those of ODC induced by hormones; (2) it is not mediated by the hormones, since adrenalectomy or hypophysectomy did not significantly modify the induction of ODC in rat liver by the drug (Cavia and Webb, 1972a; Ono et al., 1973; Suzuki et al., 1973); ( 3 ) it is quantitatively greater by far than the ODC induced by several hormones active on the liver, so that ODC has been commonly purified extensively from thioacetamide-treated rats (Ono et al., 1972). In fact, the marked elevation of liver ODC produced by the drug is roughly comparable to that observed during the earliest phases of rat liver regeneration (Fausto, 1969). Obviously, enzyme enhancement of this degree is accompanied by increased hepatic concentrations of putrescine and spermidine, but spermine concentration decreases (Fausto, 1970; Raina and Janne, 1970b). Other thioamide derivatives, such as acylthioureas, are able to induce hepatic ODC in vivo in the rat (On0 et al., 1973). In addition, the livers of rats with ODC induced by injection of thioacetamide contain multiple forms of the enzyme (Obenrader and Prouty, 1977a). Lastly, treatment with thioacetamide greatly prolongs the apparent half-life of ODC activity (Obenrader and Prouty, 197713; Poso et al., 1978) and also apparently stabilizes the enzyme, as revealed by markedly reduced sensitivity of the enzyme to inhibition by cycloheximide or 1,3-diaminopropane. Both these phenomena have also been observed for SAMD activity, i.e., lengthening of the SAMD half-life (Poso et al., 1978) and decreased sensitivity of SAMD activity from thioacetamidetreated rats to inhibition by some drugs (Poso et al., 1978). Similarly, a single treatment with another hepatocarcinogen, i.e.,

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carbon tetrachloride, induces ODC and SAMD activities in rat liver and stabilizes both the enzymes, since the inhibition of these enzymes by cycloheximide was less in animals treated with CCl, than in controls (Holtta et al., 1973; Poso et al., 1978). The time course of ODC induction by CC1, is roughly similar to that observed in thioacetamide-treated animals (Holtta et al., 1973). An accumulation of putrescine also occurs in the liver after treatment with carbon tetrachloride (Holtta et al., 1973). It should be noted that both these drugs, i.e., thioacetamide and CC14, are powerful hepatotoxic agents, so that it is quite difficult to assess how much of the ODC induction is connected with a specific effect of the drug on the enzyme and how much is a secondary effect due to the regeneration after liver cell destruction. Both the aforementioned drugs have another common effect on polyamine metabolism in the livers of treated animals. They very greatly stimulate the conversion of spermidine to putrescine, which is usually quantitatively negligible in untreated livers (Holtta et al., 1973). This effect is due most probably to a substantial increase in spermidine acetylation, which has been observed in liver extracts of rats pretreated with either carbon tetrachloride (Matsui and Pegg, 1980a) or thioacetamide (Matsui and Pegg, 1980b; Seiler et al., 1980~). These results further support the concept that monoacetylation of spermidine is a prerequisite for its conversion to putrescine, since, as previously mentioned in the introductory section, N'-acetylspermidine can be attacked by polyamine oxidase to yield putrescine. Accordingly, a single treatment with thioacetamide produced delayed increases (compared with that in ODC) in both polyamine oxidase activity and acetylpolyamine deacetylase activity (Seiler et al., 1980~). There is a wide variety of carcinogenic molecules and tumor promoters which, when administered in uiuo, specifically induce rat liver microsomal enzymes responsible for their metabolism and activation. These drugs have a common inductive pathway, which consists of the following events: (1)an increase in CAMPcontent and activation of the type I CAMP-dependent protein kinase, (2) ODC induction, and (3) induction of the specific microsomal drug-metabolizing enzymes. This sequence has been well established for rat liver after a single administration of 3-methylcholanthrene (Russell, 1971; Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). The specificity of the aforementioned biochemical sequence has been elegantly demonstrated in an inbred strain of mice, called aryl hydrocarbon nonresponsive because hepatic aryl hydrocarbon hydroxylase (i.e., the niicrosomal monooxygenase enzyme

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metabolizing polycylic hydrocarbons) cannot be induced in this strain by the administration of polycyclic hydrocarbons. 3-Methylcholanthrene injected into these animals was unable to induce any significant increase in the activities of CAMP-dependent protein kinase, ornithine decarboxylase, or RNA polymerase I (Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). Recently, we have gained further insight into the genetic control of the induction of numerous enzyme activities (including ODC) in the livers of rodents by polycyclic aromatic compounds. Nebert et al. (1980) have elegantly demonstrated that there is a strong association between the Ahb regulatory allele and ODC induction by 3-methylcholanthrene and other noncarcinogenic xenobiotics. The presence of a cytosol receptor for polycyclic aromatic inducers in genetically responsive (Ahbfihb)and heterozygotic (Ahb/Ahd)mice is essential for the process of enzymic induction. On the contrary, the genetically nonresponsive (Ahd/Ahd) mice have-as a result of a mutation-an altered cytosol receptor with a very low affinity for the inducing compounds. This cytosol receptor is therefore the major regulatory gene product. Probably, the interreaction between polycyclic aromatic compounds and a single cytosol receptor initiates the sequential induction of a series of enzymes, which includes two forms of cytochrome P-450, microsomal UDP glucuronosyltransferase, NAD(P) : menadione oxidoreductase, and ODC (Nebert et al., 1980). The Ah locus thus seems to include at least two regulatory genes, five structural genes, and perhaps one temporal gene (Nebert et al., 1980). AhblAhb-and Ahb/Ahd-responsivemice, which have suitable amounts of the cytosol receptors, respond to the polycyclic aromatic inducers with high ODC induction. Moreover, the peak in ODC activity seems to precede the peak of stimulation of total cellular RNA synthesis, which in turn precedes the peak of aryl hydrocarbon hydroxylase induction (Nebert et al., 1980). Hepatic ODC activity was induced by GH to similar extents in both Ah-responsive and Ah-nonresponsive inbred strains (Nebert et al., 1980). However, in disagreement with the results cited earlier (Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979), Nebert et al. (1980) found 3-methylcholanthrene to be without effect on hepatic CAMP-dependent protein kinase activity. A single injection of either 3,4-benzopyrene7a well-known carcinogen, or phenobarbital, a tumor promoter (Diamond et al., 1980), has been shown to induce hepatic ODC activity in rats and mice (Russell, 1971; Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). Phenobarbital is

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known to behave quite peculiarly as a promoter of hepatocarcinogenesis; it enhances the neoplastic development if given after short-term treatment with suitable hepatocarcinogens, but, if given prior to or together with the hepatocarcinogens, it inhibits tumor development in the liver (Wattenberg, 1978; Diamond et al., 1980). Phenobarbital, like 3,4-benzopyrene, is also known to induce an increase in the hepatic microsomal mixed-function oxidase system, which metabolizes it (Wattenberg, 1978). Therefore, phenobarbital has been shown to induce the same sequence of biochemical events previously described for 3-methylcholanthrene, including the induction of ODC activity, even in mice defective in aryl hydrocarbon hydroxylase (Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). Phenobarbital, 3-methylcholanthrene, and 3,4-benzopyrene were also shown to cause an enhancement of hepatic SAMD activity in the rat, but this induction occurs later than that of ODC activity (Russell, 1971). Again, as with other carcinogens, the effects of a single carcinogenic dose of diethylnitrosamine (DENA) were a rapid increase in the hepatic CAMP-dependent protein kinase, followed by ODC induction (Olson and Russell, 197913). However, a peculiarity after such a treatment was that hepatic ODC activity remained elevated for many days, a response which has never been seen after injection of any of the physiological growth stimuli (Olson and Russell, 1979b). The modifications in hepatic polyamine levels caused by a single injection of a hepatocarcinogen have been very little investigated. Single treatments with 2-acetylaminofluorene (2-AFF) or DL-ethionine increased spermidine concentration in rat liver (regardless of the sex of the animals), whereas a treatment with 3’-methyl-4-dimethylaminoazobenzene (3’-Me-DAB) had the opposite effect, decreasing spermidine and spermine contents (Raina et al., 1964; Neish, 1967). Great increases in hepatic concentrations of putrescine, monoacetylputrescine, spermidine, and N’-acetylspermidine were observed at different times after injection in rats treated only one time with thioacetamide (Raina and Janne, 1970b; Seiler et al., 1980~). Like the liver, the urinary bladder of rodents is susceptible to a variety of chemical carcinogens. ODC and SAMD activities of urinary bladders of mice or rats were induced b y topical or p.0. adminis(FANFT) or tration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), which are potent bladder carcinogens for rodents (Matsushima et al., 1979; Matsushima and Bryan, 1980). It was also observed that (1)nearly 80% of the stimulated ODC activity was located in the bladder epithelim, i.e., the cellular

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elements susceptible to carcinogenesis; and (2) the induction of ODC activity was greater than that of SAMD activity and occurred sooner after the administration of the drug than did ODC induction by hepatocarcinogens in liver (Matsushima and Bryan, 1980). As for the relationships between the chemical structure of a molecule and its ability to induce ODC, a study of a variety of 5-nitrofuran analogs demonstrated that the nitro group is essential for inducing the enzymic activity (Ertiirk et al., 1980). In studies on the connections between the synthesis of di- and polyamines in various kinds of chemically induced neoplastic growth, mammary carcinomas of female rats are one of the experimental models of choice. This neoplasm was induced by a single dose of 7,12dimethylbenz(a)anthracene (DMBA), and polyamine biosynthesis and content in the neoplastic tissue were investigated (Andersson et al., 1976). The mammary tumors showed greater ODC activity than normal mammary glands. Surprisingly, this enzyme enhancement was not accompanied by a concomitant increase in the product of this enzymatic reaction, i.e., putrescine. Of the main polyamines, the levels of spermidine and spermine, but not of putrescine, were by far higher in neoplastic tissue than in the normal control (Andersson et al., 1976). Colon carcinogenesis can be obtained by administering Nmethyl-N’-nitro-N-nitrosoguanidine(MNNG). Intrarectal instillation of this drug resulted in a dramatic and rapid induction of colonic ODC and SAMD activities (Takano et al., 1980). Very interestingly, the instillation of some physioIogica1 compounds, such as the bile salts, which are strongly suspected of having a promoting effect in colon carcinogenesis (Diamond et al., 1980), also resulted in marked ODC stimulation (Takano et al., 1980). Finally, as for polyamine catabolism in chemically induced tumors, the route of acetylation of putrescine was investigated in rat gliomas induced by a single injection of nitrosoethylurea (Seiler et al., 1975).A dramatic increase in putrescine acetylase activity and an increase in putrescine concentration were found in neoplastic tissue (Seiler et al., 1975).

B. EFFECTS O F REPEATED OR PROLONGED ADMINISTRATIONOF A CARCINOGEN OR TUMORPROMOTER ON POLYAMINE BIOSYNTHESIS AND CONTENT I N THE TARGETTISSUES Determining the polyamine content and the activities of the bios ynthetic enzymes involved after chronic treatment with a carcinogenic agent or, even better, monitoring all of them during tumor

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development helps to establish which are early derangements during neoplastic transformation, which are later ones that occur only in the developed neoplasm, and whether all or some of these derangements are characteristic of the neoplastic growth. In the rat, the changes in polyamine metabolism and content in the liver have been investigated after repeated administration of the widely used ethionine or diethylnitrosamine and during continuous feeding of 4-dimethylaminoazobenzene (DAB). In the livers of ethionine-treated rats, spermidine decreased transiently during the first days of treatment, gradually increased in the following weeks, and was still high after 6 months (Raina et al., 1964; Wainfan et al., 1978). Large doses of ethionine caused a marked fall in hepatic spermine content (Raina et al., 1964), although this was not confirmed in a later study (Wainfanet al., 1978).It is difficult to explain why these changes in hepatic polyamine patterns should be caused by ethionine, which is a well-known antimetabolite to methionine and is transformed into S-adenosylethionine. Daily treatment for several days with noncarcinogenic doses of diethylnitrosamine caused an activation of CAMP-dependent protein kinase and subsequent induction of ODC in rat liver (Olson and Russell, 197913). The extent of the ODC induction was found to be proportional to the length of treatment (Olson and Russell, 1979b). The modifications of polyamine biosynthesis and content during the hepatocarcinogenesis in rat liver due to DAB feeding have been very carefully studied and monitored by two teams almost simultaneously (Perin and Sessa, 1978; Scalabrino et al., 1978). The derangements of the polyamine biosynthetic enzymes were shown to display peculiar time courses. The activities of both polyamine biosynthetic decarboxylases, i.e., ODC and SAMD, showed marked increases as early as 1month after commencement of the dye-containing diet (Scalabrino et al., 1978). However, the increase in ODC activity was of a greater order of magnitude than that of SAMD. These increases in both hepatic enzymic activities were transient, and both enzyme activities markedly decreased during the next 2 months on the oncogenic diet (Scalabrino et al., 1978). Thereafter, a second increase in the enzyme activities occurred in the third and fourth months of azo-dye feeding, and both ODC and SAMD remained at elevated levels during the last period of the experiment. These biphasic time courses of ODC and SAMD activities, with two peaks, appear to be specific both for the liver and for the enzymes, since (a) the time course of the activities of the same enzymes in the kidneys of the same animals on the azo-dye diet from which the livers were taken were completely different from

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those in the livers and (b) hepatic tyrosine aminotransferase, an easily inducible enzyme with a very short half-life (i.e., an enzyme with some important biochemical properties in common with the two polyamine biosynthetic decarboxylases), did not display any biphasic response like that observed for ODC and SAMD in liver during azodye hepatocarcinogenesis (Scalabrino et al., 1978). The activities of spermidine and spermine synthases were constantly significantly lower in the livers of rats fed the oncogenic diet than in the control animals, the only exception being an increase in the activity of spermidine synthase at the beginning of the treatment (Scalabrino et al., 1978). As for the polyamine concentrations in rat liver during DAB carcinogenesis, the changes in putrescine concentration roughly followed the fluctuations in ODC activity, and the levels were always higher than in the controls through the entire period of experimentation (Perin and Sessa, 1978; Scalabrino et al., 1978). However, quite contradictory results have been reported for hepatic spermidine and spermine concentrations, since the significant increases in both of these polyamines observed by Perin and Sessa (1978), particularly in the last part of the experimentation, were not found by Scalabrino et al. (1978),who described decreased spermidine and spermine concentrations in livers of animals given the oncogenic diet. The rate of protein synthesis correlated well with the concentration of total polyamines in normal livers and in the livers of rats eating the DAB diet, killed every month throughout the period of treatment (Perin and Sessa, 1978). Using 3'-methyl-DABYwhich is a derivative of DAB and has greater oncogenic power than DAB, the chronobiology of the circadian rhythm of ODC in rat liver was investigated at regular time intervals from zero time until complete tumor development (Scalabrino et al., 1981). After a transient disappearance of the ODC's circadian rhythm during the first month on the oncogenic diet, this rhythm was reestablished in the livers of the rats at 60 and 90 days and then disappeared for the next 2 months. When present, the ODC rhythm in 3'-methy-DAB-treated rats had the same daily temporal pattern as that of the controls. Conversely, in the livers of rats treated with 1-naphthylisothiocyanate (a-NIT), which causes bile duct hyperplasia but never hepatic neoplasia, circadian rhythm of ODC was never detectable after only 1 month of feeding (Scalabrino et al., 1981). Therefore, although at the end of the experimental period disappearance of the hepatic ODC circadian rhythm was common to both kinds of pathological processes (i.e., hyperplasia and neoplasia), during its development each pathological process had a different, specific, well-defined change in the chronobiological pattern of the ODC rhythm. Moreover, disappear-

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ance of the ODC circadian rhythm is not a general and constant biochemical feature of all kinds of fully developed hepatomas, inasmuch as other studies have demonstrated that this rhythm disappeared in rat Morris hepatoma 7800 but not in rat Morris hepatoma 5123-C (Pariza et al., 1976a,b) (see Section 11). The alternation of presence and absence of ODC circadian rhythm might reflect changes in the cell population during neoplastic transformation. The chronobiological differences in ODC rhythm between the group fed 3’-methyl-DAB and the group fed a-NIT could be related to the different types of proliferating cells involved in the hepatic responses to the two drugs. Moreover, it has been demonstrated that, on the whole, a complete liver carcinogen such as 3’-methyl-DAB is more active in elevating ODC activity than is a simple liver hyperplastic agent such as a-NIT (Scalabrino et al., 1981). This is in good agreement with what has been demonstrated in mouse skin, where some hyperplastic agents that promote cancer weakly or not at all generally have little stimulatory effect on ODC activity, as compared with the effects of powerful tumor promoters such as the esters of the phorbol series isolated from croton oil (see Section 111,C). When we take into consideration the effects of prolonged administration of some tumor promoters, such as phenobarbital and butylated hydroxytoluene (BHT), we note that continuous feeding of the rats with either of these two drugs, surprisingly, did not enhance at all the ODC activity in liver or in lungs (Saccone and Pariza, 1978). This is in striking contrast with what has been described in Section II1,A in regard to the stimulation of hepatic ODC activity by a single injection of phenobarbital. However, the hepatic polyamine content was modified by prolonged treatment with phenobarbital, since another study demonstrated that the RNA content of mouse liver increases proportionally with the increase in spermidine and that the enhancement of DNA content was directly proportional to the spermine increase (Seiler et al., 1969). During renal carcinogenesis in the hamster due to repeated administration of 17-P-estradio1,an increase in ODC, but not in SAMD, activity was found (Nawata et al., 1980). Chronic estradiol treatment produced increases in putrescine and spennidine content in the kidneys of the treated animals, without any significant difference in spermine level between treated and control animals (Nawata et al., 1980). In a brain tumor originally induced by weekly injections of N-nitrosomethylurea and subsequently grown in tissue culture and then inoculated into the flanks of rats, the polyamine concentrations and the activities of the polyamine biosynthetic decarboxylases were

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significantly higher at the periphery of the tumor than in the center (Marton and Heby, 1974). This correlated well with the faster growth rate of the periphery than of the center of the solid tumor, as demonstrated by a markedly lower central mitotic index (Marton and Heby, 1974). C. MULTISTAGE CARCINOGENESIS Animal experiments on chemical carcinogenesis have clearly indicated that cancer development is usually a multifactorial and multistep process. Mouse skin tumors induced by aromatic polycyclic hydrocarbons are one of the most useful experimental models for studying cancer formation in uiuo. Mouse skin is certainly one of the best defined systems for studying multistage carcinogenesis. One can clearly see at least two distinct phases during chemical carcinogenesis: initiation and promotion. These two steps can be elicited by different groups of compounds (e.g., aromatic polycyclic hydrocarbons act as initiators and phorbol esters as promoters). The reader can consult the excellent reviews for details on the concept of two-stage carcinogenesis and tumor promotion, particularly in mouse skin (Boutwell, 1964, 1974; Berenblum, 1974, 1975; Scribner and Suss, 1978; Diamond et al., 1980). Here, it seems to us to be enough to recall the concepts fundamental to this topic, which are propaedeutic for understanding the derangements in polyamine biosynthesis engendered in the target tissues by this kind of carcinogenic process. A tumor promoter is a specific type of cocarcinogen that is effective only when administered after initiating action with a carcinogen given at noncarcinogenic dosages. When the sequence of the administration of the two drugs is reversed, neoplasms do not arise. In the classical model of two-stage carcinogenesis, a low, subcarcinogenic dose of a carcinogen (the initiator) is applied to mouse skin. Frequent and repeated applications of the promoter follow. Eventually, papillomas arise, but if the treatment with the promoter is further prolonged, carcinomas develop. The initiating action of the carcinogen is irreversible, whereas the effects of the promoter, which is not itself carcinogenic, are reversible. If treatment with the promoter is too meager or infrequent or discontinued too soon, tumors will not develop. Initiation could be viewed as the process of induction of some neoplastic “dormant” cells capable of being “reawakened,” whereas promotion is the process producing the appearance of the tumor and its subsequent growth (Berenblum, 1974, 1975). The conversion by the initiator of a normal cell into a “dormant” tumor cell is essentially an irreversible

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process, and a delay in the interval between completion of initiating action and the start of promoting action could not seriously affect the incidence of tumors but merely cause a corresponding delay in the time of their appearance (Berenblum, 1975). Scribner and Suss (1978) suggest that the minimal effect of an initiator is to produce isolated cells which are phenotypically identical to their unaffected neighbors with regard to functional proteins and membrane structure; these cells differ only in their ability to express altered information upon extended exposure to a promoting influence. Since only cells already transformed by initiating action can be promoted, reversal of the procedure, i.e., applying the promoting agent before the initiating agent, is ineffective in producing tumors (Berenblum, 1975). There are some conceptual points involved in two-stage carcinogenesis that are controversial and need, therefore, to be discussed briefly. First, since mutagenesis is often but by no means mandatorily connected with the carcinogenic process, the idea that initiation equals mutagenicity is quite shaky. Many strong mutagens have quite poor initiating potency (Scribner and Suss, 1978). Second, according to a former misconception, there was a prevailing tendency to consider “ cocarcinogenic action” and “promoting action” as synonymous terms, but this has been corrected (Berenblum, 1974,1975).Promoting action is only one special form of cocarcinogenic action, which has a far broader connotation. In fact, a cocarcinogen is any exogenous or even sometimes endogenous factor capable of augmenting tumor induction when it is administered with or is present with a suboptimal dose of carcinogen. Further support of the clear distinction between the two concepts of cocarcinogen and tumor promoter lies in the fact that most of the cocarcinogenic molecules do not act at all as tumor promoters in the sense defined above (Berenblum, 1974). Third, inflammation does not seem to b e a critical and obligatory step in the promotion process, since, although almost all promoters are inflammatory, not all inflammatory agents are promoters (Scribner and Suss, 1978). Therefore, an important role of inflammation in the sequential events in tumor promotion appears quite unlikely. Fourth, the real importance of the hyperplasia of the target tissues induced very frequently by promoters as an essential part of tumor promotion is uncertain, since agents inducing extensive hyperplastic stimulation have been shown to be very weak or even inactive as promoters (Scribner and Suss, 1978; Diamond et al., 1980). Experimental two-stage carcinogenesis is not limited to the mouse skin model but has been described for other organs of rodents, such as the liver, urinary bladder, colon, thyroid, kidney, lungs, mammary

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glands, stomach mucosa, and haematopoietic tissue (Berenblum, 1974; Diamond et al., 1978, 1980). Croton oil, from the seeds of the plant Crotona tiglium, has been classically employed as a tumor promoter in the mouse skin model. The promoting action of croton oil and TPA is effective for the skin of the mouse and rat, but not that of the guinea pig or rabbit. The biologically active components of croton oil are some diesters of the diterpene alcohol, phorbol. Phorbol itself is inactive in mouse skin, but, when administered intraperitoneally, is a promoter of leukemia and neoplasias of the lungs, liver, and mammary glands in rodents that have received an initiating dose of a carcinogen (Diamond et al., 1980). The relative promoting potencies of the various phorbol diesters in mouse skin are connected with their molecular structures, such as the following: (1)the steric arrangement of the phorbol molecule; (2) the presence of a free primary allylic hydroxyl group at C-20; (3)the presence of an a-@unsaturated keto group at (2-3; (4) the types of fatty acids esterified at the 12 and 13 positions; ( 5 ) among the phorbol12,13-diesters, two major groups have been identified, i.e., the A series, with a long-chain fatty acid on C-12 and a short-chain fatty acid at C-13, and the B series, with a long-chain fatty acid at C-13 and a short-chain fatty acid at (2-12, (6) esterification at C-20 or oxidation to either the aldehyde or acid at the same 20 position and the loss of the hydroxy group greatly reduce the promoting potency of the molecule; (7) the promoting activity ofthe diesters increases with increasing chain length up to 8 carbons, but, beginning with the 12-carbon length, activity markedly decreases and progressively disappears with further increasing chain length; (8)the proper steric configuration at the ring junction of C-4 and C-10 is essential for a high promoting activity of the phorbol diesters; the C-20 hydrogen and C-4 hydroxyl must be trans to maintain biological activity; when they are cis, there is great or total loss of biological activity (Fujiki et al., 1980);(9) in general, a powerful tumor promoter must have both highly lipophilic and hydrophilic portions of the molecule (Diamond et al., 1978, 1980). Other different types of diterpene esters with tumor promoting properties in mouse skin have been isolated from plants other than Crotona tiglium. Moreover, in addition to the diterpene esters, many other compounds with tumor-promoting activity in mouse skin and with a great variety of chemical structures have been identified (reviewed by Diamond et al., 1980). Among all the phorbol diesters, 12-0-tetradecanoyl-phorbol-13acetate (TPA) has been shown to have the highest tumor promoting activity in the mouse skin model. In fact, its biological and biochemi-

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cal effects were first noted and then extensively investigated in this experimental model. The structures of phorbol and TPA are shown in Fig. 6. Even a single topical application of one of the potent phorbol esters induces myriad cellular responses, both biological and biochemical. Therefore, one of the problems in the study of the mechanism of promotion has been how to distinguish between those biochemical effects elicited by the promoter that are essential for the promotion process and those effects that are collateral. Here, we will review the studies that suggest an important role of polyamines in the tumor promotion process, carried out with mouse skin, rat liver, and cell

COMPOUND

Phorbol Phorbol- 12,13- d i e s t e r s 1PA

SUBSTITUE NTS A1

12

H Fatty acid Tetradecanoate

F a t t y acid Acetate

H

FIG. 6. Structures of phorbol and its biologically active derivatives, phorbol-12,13diesters, in which the five- and seven-membered rings of the tigliane moiety are trans interlinked.

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cultures. For further details on the cellular morphological changes and the biochemical effects other than those induced by tumor promoters on polyamine biosynthesis, some excellent reviews can be consulted (Boutwell, 1964,1974,1976; Diamond et al., 1978,1980; Scribner and Suss, 1978). 1. I n Vivo Systems a. Mouse Skin Model f o r Two-Stage Carcinogenesis. With a systematic approach, O’Brien and Boutwell and their co-workers tested the effects of a large number of tumor promoters on polyamine metabolism in mouse epidermis and showed that the induction of ODC and SAMD activities is an essential component in the sequential biochemical events of promotion (O’Brien et al., 1975a,b; O’Brien, 1976, 1980). Inductions of these enzymes are among the earliest changes in biochemical systems known to occur in mouse epidermis after promoter treatment and before the general increase in total RNA and protein synthesis. In particular, the induction of ODC activity by tumorpromoting agents had been considered to be a phenotypic change essential for mouse skin carcinogenesis (0’Brien, 1976, 1980). A single topical application of croton oil or TPA, but not of phorbol, induces an astounding increase in the level of ODC activity in mouse epidermis (O’Brien et al., 1975a; O’Brien, 1976, 1980). ODC induction in mouse epidermis by TPA seems to be influenced by the age of the animals, since unresponsiveness of ODC to TPA was observed in newborn mice and in mice a few days after birth (Lichti and Yuspa, 1976). As had been observed when ODC activity was induced by other physiological or pharmacological agents, the increased epidermal ODC activity induced by TPA is predominantly located in the soluble fraction (Vermaet al., 1979a). Moreover, the induction of this enzyme activity by these tumor promoters is rapid and transient and follows a kinetics roughly similar to that observed during ODC induction by hormones in other organs of rodents, with a very sharp peak of activity 5-6 hr after the treatment followed by a return to control level after another 6 hr (O’Brien et al., 1975a; O’Brien, 1976, 1980). The stimulation of SAMD activity by croton oil and TPA was less pronounced than that of ODC, rising more slowly and showing a broader peak and a slower fall to the control level (O’Brien et al., 1975a; O’Brien, 1976, 1980). Obviously, the large increases in epidermal polyamine biosynthetic decarboxylases led to subsequent tissue accumulations of polyamines, especially of putrescine and spermidine (O’Brien, 1976). Stimulation of both the polyamine biosynthetic decarboxylases by tumor-promoting stimuli

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was dose dependent (O-Brien et al., 1975a; O’Brien, 1976). The effects of single topical applications of the different phorbol esters on ODC and SAMD activities of mouse epidermis correlated well with their promoting potencies (O’Brien et al., 197513). A possible correlation between the degree of the induction of ODC activity and the incidence of skin papillomas per mouse with the doses of TPA emerged when the amounts of TPA administered were increased (Verma and Boutwell, 1980a). The same correlation for skin carcinomas could be seen with the lowest, but not the highest, doses of TPA (Verma and Boutwell, 1980a). Pretreatment with cycloheximide at a fixed time interval before giving the tumor promoter, which was done at variable time intervals before killing the animals, resulted in an inhibition of ODC enhancement, whereas SAMD induction was prevented or decreased in some instances but not in others (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). This would indicate that induction of the two polyamine biosynthetic decarboxylases in mouse epidermis is regulated independently (O’Brien et al., 1975a; O’Brien, 1976). In contrast, pretreatment with actinomycin D always failed to block the enzyme responses to the promoters (O’Brien et al., 1975a; O’Brien, 1976). The apparently universal ability of promoters to cause induction of the polyamine biosynthetic decarboxylases was further indicated by the observation that other promoting compounds (such as iodoacetic acid, anthralin, and Tween 60, which are chemically quite different from the phorbol esters), when applied to mouse skin in an appropriate dose schedule, also induced ODC and SAMD activities in mouse epidermis (O’Brien et aZ., 1975b; O’Brien, 1976; Boutwell et al., 1979). In addition, the kinetics of the enzyme responses after single applications were different for each of these three compounds, and each differed from the responses to the promoting phorbol esters (O’Brien et al., 197513). Most surprisingly, the kinetics of ODC induction after multiple applications of one of these three compounds closely resembled the kinetics of the enzyme response after single or multiple TPA applications (O’Brien et al., 1975b; Boutwell et al., 1979). But, the kinetics of SAMD stimulation after multiple applications of iodoacetic acid, anthralin, or Tween 60 did not greatly differ from that observed after a single application of the same drugs (O’Brien et al., 1975b). The response of ODC appears to be specific, within certain limitations, for promoter agents, whereas SAMD can be stimulated in epidermal hyperplasia produced by either promoters or hyperplastic nonpromoter agents (O’Brien, 1976,1980; Boutwell e t al., 1979). Some

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hyperplastic agents, e.g., acetic acid and cantharidin, which promote weakly or not at all, caused very little stimulation of epidermal ODC activity (and delayed that) but at the same time intensely stimulated SAMD activity (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). A very minimal stimulation of mouse epidermal ODC was elicited by a compound such as 4-0-methyl-TPA, which is a pure hyperplasiogenic agent with very weak tumor-promoting activity (Marks et al., 1979). Therefore, the enhancement of ODC activity brought about by the promoters does not seem to be related to the stimulation of cell proliferation by these drugs, since these two biochemical and biological events, i.e., ODC induction and cell proliferation, can be suitably dissociated in mouse epidermis, as occurs with hyperplastic agents that stimulate cellular multiplication but not ODC activity. A single topical application of DMBA or other carcinogenic hydrocarbons, such as benzo [a ] pyrene, 3-methylcholanthrene, and 1,2,5,6-dibenzanthracene7at initiating doses (i.e., at low doses) to mouse skin did not at all affect epidermal polyamine biosynthetic decarboxylase activities (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). However, when the same molecules were tested at high dosages, i.e., when they acted as complete carcinogens, epidermal ODC and SAMD activities were intensely stimulated (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). Noncarcinogenic hydrocarbons were ineffectivein inducing ODC (O’Brien, 1976; Boutwell et al., 1979). This differential response of ODC activity to initiating and carcinogenic doses of a drug seems to further support the idea that ODC enhancement is strictly related to the promotion process (O’Brien et al., 197513; Boutwell et al., 1979). Most interestingly, ODC levels, though not SAMD levels, have been shown to correlate well with the degree of malignancy of mouse skin two-stage neoplasms, because papiIlomas had much lower ODC levels than carcinomas, and the values for both kinds of tumors were higher than those of normal epidermis (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). It is also worth noting here that the ODC in cutaneous papillomas, in turn, had a half-life time much longer than the half-life of ODC in normal skin (O’Brien, 1976). Conversely, the half-lives of SAMD were not significantly different in normal skin and in cutaneous papillomas (O’Brien, 1976). On the grounds of all the above, O’Brien (1976) came to the conclusion that the induction of ODC activity is at least one of the essential, possibly obligatory, biochemical events in carcinogenesis of mouse

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skin. In other words, the promotion process may cause a constant increase in the ODC activity in the initiated cells and thus provide them with a biochemical tool useful for uncontrolled and prolonged growth. A large number of later studies have further clarified some other molecular aspects of epidermal ODC induction after topical application of tumor promoters. First, the induction of this enzyme by TPA is much lower in mice maintained for a suitable time on a vitamin Bedeficient diet (Murray and Froscio, 1977). It is well known that ODC requires pyridoxal-5-phosphate as a cofactor. Complementing this, the administration of pyridoxal-5-phosphate to vitamin Be-deficient mice again allowed normal induction of epidermal ODC activity by TPA, as in the control animals (Murray and Froscio, 1977). Unlike what has been described for ODC, application of TPA induced marked and similar enhancements of epidermal cell proliferation in both mice maintained on a complete diet and mice on a vitamin B6-deficientdiet (Murray, 1978). Consequently, these results suggest that there is no causal or mandatory relationship between ODC induction and induction of hyperplasia by TPA. Second, ODC induction by TPA does not appear to be mediated by earlier increases in the epidermal levels of CAMP and/or cGMP, because a single topical application of this phorbol diester at doses inducing ODC caused no increases in the levels of either of these cyclic nucleotides (Mufson et al., 1977; Mufson, 1978; Boutwell et al., 1979; Marks et al., 1979). This confirmed once more that the regulation of ODC induction in some mammalian tissues by cyclic nucleotides is by no means a general phenomenon (see Section 1,B). However, a conflicting report has appeared showing that a single topical preapplication of 3-isobutyl-l-methylxanthine, a well-known potent inhibitor of cyclic nucleotide phosphodiesterases, before the TPA application acted synergistically with TPA in increasing ODC activity, with a smaller TPA dose needed to obtain the same or even higher values of ODC activity (Perchellet and Boutwell, 1980a). Therefore, whether cyclic nucleotides are really the second messengers for the growth-related increases in RNA and protein syntheses that occur in the epidermis after TPA application remains a moot question. Third, it has been claimed that prostaglandins El and Ez are involved selectively in the induction of epidermal ODC activity by TPA, since application of PGEl and PGE2, but not of PGFl and PGFk, with the application of TPA resulted in removal of the inhibition of ODC induction caused by pretreatment with indomethacin or acetylsalicylic acid, well-known inhibitors of prostaglandin synthesis (Verma et al., 1978). But indomethacin pretreatment did not prevent the induction of SAMD by TPA (Verma et al., 1978). Furthermore, simulta-

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neous application of PGE2, but not of PGF2, and TPA reversed the blockage of the cell proliferative response of mouse epidermis to TPA induced by pretreatment with indomethacin (Furstenberg and Marks, 1978). Therefore, unlike vitamin B6deficiency, the PGEs seem to be factors involved in controlling both ODC induction and cell proliferation caused by TPA. Fourth, since one of the routes by which ornithine is synthesized is via hydrolysis of arginine by arginase to yield ornithine and urea, the effect of TPA application on arginase activity in mouse epidermis has been tested (Vermaet al., 1979a). Arginase activity was unresponsive to such treatment, indicating that this enzyme is not a limiting step in the stimulation of polyamine biosynthesis by TPA (Verma et al., 1979a). The foregoing leading idea that epidermal ODC activity is nearly exclusively stimulated by tumor-promoting agents, whereas nonpromoting stimulators do not have this effect, has been critically revised in some recent studies. It has been demonstrated that epidermal ODC activity is strongly increased not only by TPA but also by Ti 8*, which is a TPA structural analog that has essentially no tumorpromoting power but still has mitogenic and irritant properties similar to those of TPA (Marks et al., 1979). Mezerein, too, which is a diterpene ester of plant origin, like TPA, and has many close structural similarities to TPA, has been tested for its biological and biochemical effects (Mufson et al., 1979). Mezerein, although it is equipotent with TPA on an equimolar basis in inducing hyperplasia, in inflammatory activity, and especially in inducing ODC activity in mouse epidermis, is a very weak mouse skin tumor promoter (Mufson et al., 1979). Ethylphenylpropiolate (EPP), another good inflammatory and hyperplasiogenic compound with weak tumor-promoting power, caused a marked increase in both ODC and SAMD activities in mouse epidermis after a single topical application (Takigawa et al., 1977; Boutwell et al., 1979). In this case, too, the induction of ODC activity changed in parallel with concomitant changes in skin putrescine concentrations (Takigawa et al., 1977). These authors doubt .the idea that the tumor-promoting potency of a compound can be exactly mirrored by its effect in inducing ODC activity. In order to further clarify the links, if any, between ODC induction and the tumor-promotion processes, another experimental approach has been employed in recent years that consists of the replacement of chemical tumor-promoting stimuli with mechanical ones. Cutaneous papilloma development has been “initiated” by a carcinogen mole* Ti 8; 12-0-tetradeca-2-cis,4- trans-6,8-tetraenoylphorbol-l8acetate.

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cule, i.e., DMBA, administered in a subthreshold dose; thereafter, the mechanical treatments were either skin massages (which cause cell proliferation and are devoid of any tumor-promoting power) or skin wounding (which also induces cell proliferation and is, additionally, a promoting stimulus). A marked enhancement of epidermal ODC activity was observed after skin wounding but not after skin massages (Clark-Lewis and Murray, 1978). On the other hand, TPA has been found to stimulate ODC activity in rat skin, a tissue in which it was not previously known to have been a tumor promoter (Lesiewicz et al., 1979, 1980).The ODC activity of rat skin was stimulated additively by TPA application plus hair plucking (Lesiewicz et al., 1979, 1980). The ODC stimulation by either TPA or hair plucking has been found to be much greater in the rat epidermis than in the rat dermis (Lesiewicz et al., 1980). The time course of the ODC response to TPA differs in several aspects from that to hair plucking (Lesiewicz et al., 1980). Aerosolized TPA was also shown to induce pulmonary ODC activity in mice in a dose-dependent manner (Dinowitz et al., 1980). In conclusion, one can reasonably think that all these results combined argue for the idea that mouse skin tumor-promoter stimuli, both chemical and mechanical, generally cause hyperplasia, irritation, and ODC induction, but the reverse is not true, i.e., no stimulus that elicits all of these three biological responses has necessarily to be considered a tumor promoter. Consequently, this triad of biological and biochemical reactions to the tumor promoters is nearly a constant in the tumorpromotion process but is not sufficient per se to accomplish this process. Therefore, the relevance of these and other enhancements of some cellular activities induced by the promoting phorbol esters to the mechanism of skin tumor promotion is still unsolved. Similarly, it is still a matter of speculation whether there is a causal relationship between ODC induction and the tumor-promotion process, on the one hand, or between ODC induction and cell proliferation, on the other. The particular aspects of the stimulation of polyamine metabolism in the two-stage protocol of mouse skin carcinogenesis have been reviewed by Slaga et al. (1978), Boutwell et al. (1979), Lesiewicz and Goldsmith (1980), and Lowe (1980). b. Models of Two- or Multistage Carcinogenesis in Livers of Rodents. There are very few studies available in the literature on this subject. No enhancement was seen in ODC activity in the livers of rats that received diethylnitrosamine (DENA) in drinking water and then, after discontinuation of this nitrosamine, were given phenobarbital in the diet, although phenobarbital greatly increased the incidence of liver tumors induced by DENA, i.e., was very effective as a tumor promoter for rat liver (Farwell et al., 1978). Neither was hepatic ODC

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activity induced by DENA alone or by phenobarbital alone (Farwell et al., 1978). Again, most surprisingly, no increase in ODC activity was found in fully developed hepatocarcinomas induced by DENA only or resulting from phenobarbital promotion of DENA carcinogenesis (Farwell et al., 1978). A three-stage model of carcinogenesis for rat liver has been proposed by Solt and Farber (1976). The typical sequence of such a protocol consists of a DENA injection as initiator, followed by a low level of 2-acetylaminofluorene (AFF)in the diet for a short time period, acting as a selective growth inhibitor, and finally followed by partial hepatectomy, acting as a generalized potent growth stimulus. This sequence of treatments cannot be reversed without greatly reducing the incidence of hepatic neoplasms. In this multistage carcinogenesis model, CAMP-dependent protein kinase and ODC activities were investigated (Olson and Russell, 1979a). The activities of these two enzymes were enhanced, first one and then the other, shortly after DENA administration and remained elevated for several days. After waning to the control levels, the activities of both these enzymes were stimulated once again by partial hepatectomy and remained high for two weeks thereafter (Olson and Russell, 1979a). Therefore, in this three-stage model of hepatic carcinogenesis, enhancement of ODC activity in the target organ appears to be tightly linked with the carcinogenetic process, which is quite different from what has been previously described for the two-stage model for the same organ. As in the skin protocol, one of the two stimuli in the two-stage scheme for hepatic carcinogenesis can be surgical. It is well established that rapidly proliferating rat hepatocytes, namely, regenerating liver, are more sensitive to the carcinogenic effects of urethan than resting cells. Injection of this drug has been shown to suppress to similar extents both the typical biphasic ODC enhancement and the increase in intracellular cAMP levels, both induced in the liver remnant by partial hepatectomy (Matsui et al., 1978, 1980). However, after this early suppression phase, ODC and SAMD activities again rose in regenerating livers of rats that were given urethan, i.e., enzyme inductions were only delayed in comparison with regenerating controls (Matsui et al., 1980).Unlike liver regeneration, the enhancement of hepatic ODC activity by several hormones was not suppressed and the intracellular cAMP level not modified b y administration of urethan (Matsui et al., 1978). Inasmuch as urethan is known to inhibit in vivo hepatic DNA and RNA syntheses, it must be clarified whether the parallelism between the suppression of ODC induction and the impairment of DNA synthesis expresses a cause-effect relationship and whether the inhibition of ODC activity is an obligatory step in the

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molecular mechanism by which urethan induces neoplasms. This last would represent an interesting and perhaps unique exception in ODC responses to carcinogens, since, as previously demonstrated by a large body of evidence, carcinogens generally have the ability to induce ODC activity in target organs.

2. In Vitro Systems TPA and other promoting phorbol esters have been tested for ODC induction and other effects on polyamine metabolism in different in vitro cellular systems, which can be classified into those consisting of mouse epidermal cells and those consisting of cells other than epidermal ones. Such a division is justified in order to tentatively compare the effects in in vitro systems with those in in vivo systems, although this extrapolation must be made with great caution. In fact, at present it is still questionable whether the in vitro tumor promotion process is an exact mirror of the same process in viuo. Nevertheless, all in vitro systems can generally be used as tools for investigating and dissecting the molecular mechanisms involved in tumor promotion. Certainly the mouse epidermal cell culture system appears to be by far the most appropriate for studying the promotion process in vitro, since the tumor promotion concept was first defined in the mouse skin model of carcinogenesis in vivo. a. Cultures of Mouse Epidermal Cells. The effects of a series of phorbol esters, including both those with a wide range of tumorpromoting potency and those without any, on DNA synthesis and ODC activity have been investigated in mouse epidermal cell cultures (Yuspa et al., 1976). A good correlation between the effectiveness of the phorbol esters as tumor-promoting agents in mouse skin in vivo and their ability to stimulate DNA synthesis and ODC activity in vitro was found (Yuspa et al., 1976). The kinetics of the ODC induction were roughly similar to those observed in the same cells in vivo, and this enzymic stimulation chronologically occurs much earlier than stimulation of DNA synthesis (Yuspa et al., 1976). By analogy, similar results were obtained in cultured epidermal cells from newborn mice (Lichti and Yuspa, 1976; Lichti et al., 1978a). Moreover, the induction of ODC activity by TPA in cultured mouse epidermal cells was completely prevented by the previous or contemporaneous presence of cycloheximide in the medium, indicating that the enzyme induction requires de novo protein synthesis (Lichti et al., 1978b). In contrast, actinomycin D at doses that completely inhibit total RNA synthesis paradoxically enhanced ODC induction by TPA (Lichti et al., 1978b). ODC induction by TPA in isolated epidermal cells appeared to be CAMP and cGMP dependent, because TPA treatment enhanced the

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cellular levels of both cyclic nucleotides, and the simultaneous presence of 3-isobutyl-1-methylxanthine (a well-known inhibitor of cyclic nucleotide phosphodiesterase) potentiated the ODC induction by the promoting agent (Perchellet and Boutwell, 1980a,b). In the aforementioned system of cultured cells, the magnitude of ODC induction was also dependent on the duration and manner of TPA treatment, and, what is more, it changed considerably at various times after plating, since the cells first lost and then regained their responsiveness to TPA (Lichti et al., 1978a). Furthermore, the correlation between the intensity of the stimulation of ODC activity in vitro and the potency of the tumor promoters in vivo is not a general and constant feature of tumor-promoting agents tested in uitro, since this correlation was barely observed for several Tween compounds with a wide range of tumor-promoting potencies (Lichti et al., 1978a). Unlike its effects on ODC activity, TPA treatment was found to produce a dramatic and quite unexpected fall in SAMD activity in cultured epidermal cells, which was also unlike the stimulating effect on SAMD activity observed in vivo (Lichti et al., 1978a). Further evidence of the dissociation of induction of ODC activity from stimulation of DNA synthesis, both caused by TPA in cultured mouse epidermal cells, has been provided by Hennings et al. (1978), who investigated the effects of varying cell culture conditions, such as pH and serum content of the medium, on these two biochemical parameters. TPA induced identical increases in DNA synthesis regardless of differences in serum levels during treatment, although an optimal serum level is required for maximal cellular proliferative responses to TPA. But the absence of serum in the medium greatly reduced ODC induction by TPA, thus indicating that the increase in ODC activity, which always chronologically precedes DNA synthesis, is not a necessary prerequisite for the stimulation of DNA synthesis. Again, lowering of the pH of the culture medium affected ODC induction and stimulation of DNA synthesis b y TPA to different extents, since DNA synthesis and cell proliferation were inhibited at pH 6.7 more than ODC response (Hennings et a,?., 1978). The presence of serum in the incubation medium for mouse skin explants allows much more protracted ODC induction than that observed in the same cultures in the absence of serum (Verma and Boutwell, 1980b).As for the cultured epidermal cells, the induction of ODC activity in incubated mouse skin explants is dependent on the concentration of TPA in the medium (Verma and Boutwell, 1980b). b. Cultures of Cells Other than Epidermal Cells. The relevance of the studies with cultures of nonepidermal cells reported here can be questioned, since the types of cells used are not the natural target cells

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for TPA’s promoting activity in wiuo. In cultures of hamster embryo cells (fibroblasts), a variety of tumor-promoting phorbol diesters was found to induce ODC activity (O’Brien and Diamond, 1977; O’Brien et al., 1979). The same result was found in another cell line derived from hamster embryo cells transformed malignantly b y benzo(a)pyrene. TPA caused a much greater induction of ODC activity in the transformed cells than in the normal ones, although the basal ODC levels of the two types of cell lines were quite similar (O’Brien and Diamond, 1977, 1978a, 1979; O’Brien, 1980). ODC activity was also induced by adding fresh medium to both kinds of cell line cultures, but the magnitude of the enzyme induction was greater in transformed cells than in normal ones (O’Brien and Diamond, 1977, 1978a, 1979; O’Brien, 1980). Likewise, when fresh medium and TPA were added simultaneously, the stimulating effects on ODC activity in normal cells were approximately additive, whereas they were synergistic in transformed cells (O’Brien and Diamond, 1977, 1978a; O’Brien et al., 1979, 1980). This and the earlier results suggest that ODC activity in malignant cells is more sensitive to positive stimuli, either promoting or not, than are normal cells. Other examples, besides that of ODC activity, of enhanced responses to TPA by transformed cells are known and involve morphological changes or metabolic activities, e.g., prostaglandin synthesis (the corresponding references are cited in Diamond et al., 1978). The potentiation of ODC induction by combined TPA and fresh medium appears to be specific for this enzyme, since no similar synergistic or additive effect for SAMD activity was seen in the same cultured cells, either normal or transformed, under the same experimental conditions (O’Brien, 1980; O’Brien et al., 1980). On the other hand, TPA alone induced SAMD activity in both cell types, i.e., normal and transformed (O’Brien, 1980; O’Brien et al., 1980). In transformed cells, ODC enhancement by TPA produced a parallel effect in putrescine concentration but not in spermidine and spermine (O’Brien and Diamond, 1977; O’Brien, 1980). In contrast, no significant changes in polyamine concentrations were observed in normal cells after treatment with TPA (O’Brien, 1980). Unlike ODC induction in cultured normal mouse epidermal cells, the TPA-induced increase in enzyme activity in transformed cells was completely inhibited by cycloheximide and by actinomycin D (O’Brien and Diamond, 1977, 1978a, 1979). Another difference in biological responses to TPA of cultured normal epidermal mouse cells and hamster embryo fibroblasts, whether transformed or not, is that in the latter type of cells DNA synthesis and cell division were not affected by exposure to tumor promoters (O’Brien and Diamond, 1977, 1978a, 1979; O’Brien, 1980).

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Other than in hamster embryo cells, TPA stimulated ODC activity in 3T3 mouse cells and PHA-treated bovine lymphocytes, but not in human fibroblasts or rat embryo fibroblasts (Kensler et al., 1978; O’Brien et al., 1979). Furthermore, TPA stimulated DNA synthesis only in 3T3 mouse cells and human fibroblasts (O’Brien et al., 1979). Therefore, no mandatory relationship between ODC induction by TPA and stimulation of DNA synthesis by the same molecule can be drawn. TPA loses its ability to induce ODC in both normal and transformed hamster embryo fibroblasts when it is completely converted by the cells into phorbol-13-acetateYwhich is not further metabolized and has no ODC inducing activity (O’Brien and Diamond, 1978b). Surprisingly, although some human cell lines were found to be unable to metabolize TPA, the same cell cultures were unable to induce ODC activity by TPA (O’Brien, 1977, 1980; O’Brien and Diamond, 1978b; O’Brien et al., 1979). Several recent studies have suggested that an early and possibly primary site of action of tumor-promoting phorbol esters is the cell membrane, where they might modify structure or function. Recently, it has been demonstrated that the phorbol ester tumor promoters act, at least in part, through a specific hormonal pathway, since these molecules and the neurohypophyseal hormone vasopressin stimulate some biochemical parameters of quiescent cultured mouse Swiss 3T3 cells by a common mechanism (Dicker and Rozengurt, 1980). In fact, TPA and vasopressin can substitute for each other in stimulating DNA synthesis and 2-deoxyglucose uptake and in inducing ODC activity (Dicker and Rozengurt, 1980). TPA and vasopressin added simultaneously to the culture medium show neither additive nor synergistic effects in enhancing all these biological activities. In contrast, TPA has been shown to act synergistically with some well-known growthstimulating polypeptides or hormones, such as insulin and epidermal growth factor (EGF), in stimulating some biochemical parameters (cited in Dicker and Rozengurt, 1980). However, the combination of TPA and insulin was synergistic for ODC induction in chemically transformed hamster embryo fibroblasts, but not in hamster fibroblasts (O’Brien et al., 1980). A very important question, as yet not fully understood, is whether removal of regulation from polyamine biosynthesis could be a key difference between neoplastic transformation and normalcy. Besides the differences in polyamine metabolism between normal and transformed cells previously reported, support for this idea has been provided by O’Brien et d . (1980), who demonstrated that there is normal production in untransformed hamster fibroblasts of the ODC antizyme elicited by putrescine, whereas there was little or no production in

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transformed cells similarly treated with putrescine. This difference in ODC antizyme production occurred despite nearly equal sensitivity of the ODC activities of both cell lines to the inhibitory effects of exogenous putrescine on the enzyme activity (O’Brien et al., 1980). The polyamine metabolic abnormalities plus other metabolic derangements induced by tumor-promoting phorbol esters (especially TPA) in different cell lines in vitro have been reviewed by Diamond et al. (1978, 1980) and O’Brien (1980). To summarize, some major points should be recalled and stressed here : 1. From the results so far reported, it is quite clear that there is no one unique and common behavior of polyamine metabolism after the different phorbol ester treatments in uitro. This is not at all surprising and is in perfect agreement with a large body of experimental evidence that illustrates the great diversity of biological effects produced by promoters in different in vitro systems. Therefore, no general rule valid for every in vitro system and for the effects of every tumorpromoting agent can be drawn. Consequently, the exact role played by polyamines in the tumor-promotion process in vitro is still a matter of speculation. Frequently, however, some similarities in the responses to phorbol esters observed in comparable in vitro systems can be seen and tentatively interpreted. 2. TPA and related compounds induce a large number of biological alterations in cell cultures that mimic those often associated with cell transformation by chemical carcinogens or oncogenic viruses (Diamond et al., 1978). 3. The expression of certain cellular “markers” of transformation in phorbol ester-treated cultures depends on the continuous presence of the agent, so that the normal cells exposed to a promoting phorbol ester can revert to their previous phenotype once the agent is removed. Clearly, this is in striking contrast to the situation in malignant cells, in which the expression of these properties is autonomous. 4. When the biological actions of initiating carcinogens (usually complete carcinogens by themselves) and promoting agents are compared, the most striking difference lies in the fact that initiators can yield electrophil and bind covalently to cellular macromolecules, such as proteins and nucleic acids, whereas there is, up to now, no evidence that promoters bind covalently to these macromolecules. 5. The possibility that phorbol esters have completely different effects in different tissues and species, converting and recognizing the

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initiated cells to neoplastic cells in one species or tissue but not in another, cannot be ruled out. 6. The importance of the studies with two- or multistage carcinogenesis models is not at all limited to the experimental field, since there is ever-increasing evidence that tumor-promoting agents, known or unknown, in the environment may be factors that contribute to the induction of tumors in human beings.

D. MUTAGENICACTION A N D ANTIMUTAGENIC PROPERTIES OF POLYAMINES

1. Mutagenic Efects of Nitroso Derivatives of Spermidine and Spermine It is well known that the “mutation” theory and the “aberrant differentiation” theory have long been discussed as separate mechanisms for carcinogenesis; recently, the “mutation” theory has received more support, since most carcinogens have been found to be mutagens. The ability of carcinogens to bind to DNA and chromatin, resulting in altered structure and function of genetic material, can easily explain their mutagenicity. In this respect the finding that nitrosated spermidine has mutagenic properties in a biological assay system utilizing Salmonella typhimurium is of great interest (Kokatnur et al., 1978; Hotchkiss et al., 1979). Both spermidine and spermine contain secondary and primary amine groups which can be nitrosated by nitrite. Nitrosation can occur during the digestion of numerous foods containing large amounts of spermidine, spermine, and nitrite. Nitrite is produced in human saliva, and its involvement in N-nitroso compound formation in the gastrointestinal tract is reasonable to suppose. It is well known that the most recent views on the etiology of human gastric cancer have focused on the hypothesis that some N-nitroso compounds formed in the stomach are directly responsible, as mutagenic carcinogens, for this type of neoplasia. A variety of N-nitrosamines which arise from nitrosation of spermidine, many of which have been demonstrated to be or are strongly suspected to be carcinogenic, have been identified and characterized (Hildrum et al., 1975; Hotchkiss et al., 1977). Some of these products are volatile, some are not. The mutagenic action of nitroso derivatives of polyamines and the involvement of these products in the etiology of cancer has recently been reviewed (Murray and Correa, 1980).

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2. Antimutagenic Properties of Spermidine and Spermine Spermidine and spermine, because of their basic nature, bind noncovalently to the phosphate residues of DNA and neutralize their negative charges. Thus, the ordered structure assumed by DNA in the presence of spermidine prevents the intercalation of 3,4-benzo[a] pyrene into DNA (Liquori et al., 1967). Spermine, on the other hand, again because it binds to the minor groove of DNA and subsequently stabilizes DNA structure, inhibits the methylation of both chromatin DNA and free DNA induced by N-methyl-N-nitrosourea (MNU), a well-known and potent carcinogenic and mutagenic agent, in eukaryotic cells (Rajalakshmi and Sarma, 1978; Rajalakshmi et al., 1978a,b). It is not known at present whether the protective effects of spermidine and spermine against carcinogen-DNA interaction are limited to methylating agents, such as MNU, and polycyclic hydrocarbons, such as 3,4-benzo[a]pyrene or can be extended to other types of carcinogens, The antimutagenic properties of spermine and other polyamines in microbial systems have been reviewed (Clarke and Shankel, 1975).

E. POLYAMINE LEVELSIN URINE, SERA, AND ERYTHROCYTES OF

RATS DURING CHEMICAL CARCINOGENESIS OR BEARING SEVERAL EXPERIMENTAL TUMORS

As has been done in humans with different types of neoplasms (see Section 11, Part I1 of this chapter in Vol. 36), the urinary polyamine levels in experimental animals undergoing chemical carcinogenesis or acute treatment with carcinogens have been monitored by several authors. A significant increase in urinary putrescine only was observed in mice which had received a single carcinogenic dose of 3,4-benzo[al pyrene given subcutaneously (Fujita et al., 1978).I n more detail, the urinary putrescine level began to be significantly increased 2 months after the 3,4-benzo[ alpyrene administration, then became even more pronounced 1 month later, reaching a maximum at the fourth month (Fujita et al., 1978). Significant increases in the urinary levels of total polyamines, as well as of each of the chief polyamines in DABhepatoma-bearing rats have been recorded, but the increases of putrescine levels were less than those of spermidine and spermine (Perin and Sessa, 1978). In rats with mammary carcinomata induced by DMBA, only the 24-hr urinary excretion of putrescine was found to parallel tumor

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growth and mass (G. Andersson et al., 1980). But, when the neoplasm was in the final stages of its progression, the correlation disappeared, since the urinary concentration of putrescine decreased unexpectedly (G. Andersson et al., 1980). In a previous study, other authors found only slight increases in the urinary excretion of putrescine and spermidine in rats with the same type of neoplasm induced by the same carcinogen (A. C. Andersson et al., 1976). In the urine of rats with experimental gastric tumors induced by chronic administration of N-methyl-N-nitroso-N’-nitrosoguanidine, there was a significant and early elevation in the putrescine level that became even more marked during the final phases of gastric carcinogenesis (Fujita et d., 1976). In the urine of rats that had received a subcutaneous implant of an immunocytoma, a statistically significant circadian rhythm was apparent for the excretion of putrescine, spermine, and cadaverine, but not for spermidine (Halberg et al., 1976). At all six time points of the day, the rate of excretion for all four polyamines was higher in tumorbearing rats than in control rats, with the overall rate being about twice as great as in controls (Halberg et aZ., 1976). As for serum, rats with chemically induced brain tumors transplanted into flanks had serum spermidine concentrations markedly higher than the serum values of control animals (Marton and Heby, 1974). The same was found in rats with mammary tumors (Russell et al., 1974b,c; Russell and Durie, 1978). When this tumor regressed after castration and hypophysectomy, the serum level of spermidine behaved bimodally, first increasing and then decreasing (Russell et d., 1974b,c; Russell and Durie, 1978). The increase in the serum paralleled an analogous trend of the spermidine levels in the tumor’s interstitial fluid (Russell et al., 1974b,c; Russell and Durie, 1978).These data suggest that the elevated level of spermidine in the sera of rats with tumors in regression phase is mainly a result of spermidine release from the tumor tissue, due to cell death. In further support of this interpretation, the same bimodal time course was observed for the spermidine level in the sera of rats with a Morris hepatoma after effective antineoplastic chemotherapy (Russell et al., 1974a,d; Russell and Durie, 1978). Again, shortly after a single injection of the antineoplastic drug used, 5-fluorouracil, the spermidine concentration within the tumor markedly decreased owing to a marked decrease in the number of hepatoma cells, just at that time when the spermidine concentration in the serum doubled (Russell et al., 1974a,d; Russell and Durie, 1978). By studying the polyamine levels in the sera of rats with this same tumor after local radiation, a rapid increase in spermidine was

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once again seen, but an unexpected similar change in putrescine level was found concomitantly (Russell et al., 1976b; Russell and Durie, 1978).As was observed after chemotherapy, the spermidine concentration within the hepatoma rapidly decreased after local radiation (Russell et al., 1976b; Russell and Durie, 1978). Other studies have carefully investigated the importance of the cellloss factor of a neoplasm in determining elevations of or modifications in the levels of the extracellular polyamines in the sera of animals with different types of tumors. Actually, the concentrations of putrescine and spermidine in cell-free ascites fluid and serum of mice with Ehrlich ascites tumor cells were found to increase greatly not only during tumor growth but also when there was a considerable decrease in the rate of cell proliferation (Heby and Anderson, 1978). That tumor cell death is the cause of increased polyamine levels in physiological fluids in cancer has been cleverly confirmed by inducing tumor cell death by heterologous transplantation of the same type of mouse tumor into gerbils and by following the tumor cell loss (G. Anderson et al., 1978a; Heby et al., 1978a).The concentrations of polyamines in cell-free ascites fluid and serum showed patterns similar to those of the activity of tumor lactate dehydrogenase in the same fluids, the peaks of the time courses for polyamines and lactate dehydrogenase being closely coincident with the time of maximal tumor cell death (G. Andersson et al., 1978a; Heby et al., 1978a). As in patients with malignant disease, polyamine levels in peripheral erythrocytes have been measured in animals with different types of neoplasms. A positive correlation between spermidine and spermine levels in red blood cells of mice with Ehrlich ascites carcinoma cells and the tumor growth has been established, since the increases in the levels of these two polyamines occurred very soon after i.p. inoculation of the ascites carcinoma cells (Uehara et al., 1980). By analogy, erythrocyte spermidine concentration increased with increasing tumor mass in rats transplanted subcutaneously with a fast-growing solid hepatoma (Shipe et al., 1980; Wills et al., 1980). In contrast, in this situation erythrocyte spermine concentration did not at all reflect increases in tumor mass (Shipe et al., 1980). The levels of the main polyamines have been compared in plasma and in red blood cells of mice transplanted with melanoma (Takami and Nishioka, 1980). The levels of all the measured polyamines in both erythrocytes and plasma rose as the number of days after tumor inoculation increased (Takami and Nishioka, 1980). However, the levels of putrescine, spermidine, and spermine in erythrocytes showed progressive increases much greater than those in plasma (Takami and Nishioka, 1980).

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Mathematical models have been formulated to relate tumor cell number to intracellular polyamine concentrations and, utilizing the pharmacokinetics, to predict the polyamine compartmentalization between plasma and organs in which neoplasms have been developed (Woo and Simon, 1973; Himmelstein et al., 1976; Woo and Enagonio, 1977; Himmelstein and Rosenblum, 1980). From all the results reported here concerning the polyamine levels in the urine, the fluid part of blood, and/or the erythrocytes of experimental animals with different types of neoplasms, no single, clear, unique indication has emerged about whether or not an increase of one or more polyamines in the physiological fluids or cells is of any real help in evaluating the growth rate of the experimental tumors. Therefore, the value of polyamine determinations in these biological compartments as a tool for diagnosis or prognosis of cancer remains questionable. One must also keep in mind that high polyamine levels in physiological fluids and/or erythrocytes of experimental animals have been observed in nonpathological conditions, such as during pregnancy and lactation or after hormone administration in the rat (A. C . Andersson et al., 1978b; Lundgren and Oka, 1978; Rojansky et al., 1979; Andersson and Henningsson, 1980b) or in mice after intraperitoneal injection of bovine serum albumin (Uehara et al., 1980). These conclusions on the nonspecificity of the increases of polyamine levels for tumors and on their promise for evaluating the effectiveness of antineoplastic therapy are in keeping with what will be stressed and discussed in the section on human oncology (see Section 11, Part I1 of this chapter in Vol. 36). IV. Biosynthesis and Levels of Polyamines in Cells during the Virus-Induced Transformation Process

Oncogenic viruses are not only of great importance in the etiology of some animal tumors, but some human tumors are also associated with and actually strongly suspected to be caused by viruses. Since the original demonstration by Rous of sarcoma induction by a chicken virus, two distinct classes of cancer-causing viruses have been identified in a range of vertebrate species. Some are DNA viruses and others RNA viruses. Among the DNA viruses of vertebrates, some members of the papova-, adeno-, herpes-, and poxvirus groups are known to be oncogenic for different animal species. Among the RNA viruses, all known oncogenic viruses belong to one group, i.e., the retraviruses, also called retrovirus or oncornavirus (Fraenkel-Conrat, 1974).

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A normal cell population is transformed by infection with an oncogenic virus, followed by isolation of colonies of cells with altered properties. Alterations are found in cellular morphology, there is piling up of cells that normally appear to grow as monolayers, and there are a myriad of biochemical changes at both the surface and the nonsurface level of the cell. Therefore, transformation is defined operationally; it is useful for the identification of virus-induced changes in cultured cells, but it is not a universal correlate of oncogenicity. Most DNA tumor viruses and the sarcoma-inducing retroviruses cause cellular transformation, while leukemia-causing oncornaviruses usually cause cell growth without transformation, with some exceptions. For this reason, the leukemia viruses are often called “nontransforming viruses’’. Furthermore, many of the properties used to define transformation can be transiently induced in normal cells by experimental manipulations, e.g., treatment with proteolytic enzymes. Tumor viruses are obviously defined by their capacity to induce neoplasms in animals. By the same token, transformed cells are neoplastic only if they can grow into tumors in an appropriate host. Nonetheless, although cancer is a disease definable only in whole animals, an analog of malignancy called “cellular transformation” provides an in vitro model on which almost all work with oncogenic viruses is based. In fact, the phenotypic changes which serve to identify transformation of cultured cells are indispensable aids in the investigation of tumor viruses. The normal cells used for transformation studies generally are of two types: cells taken from embryos, often from chicks or rodents, and permanent lines of mammalian cells that can be cloned. Experimental models that involve the use of oncogenic viruses have been devised to test the correlation of polyamine synthesis with neoplastic growth. These models have definite advantages, stemming from the rapidity of transformation and from the ability to perform kinetic experiments with normal and malignant cells derived from the same source and grown under well-controlled and identical experimental conditions. In this section, the changes in polyamine biosynthesis and content of different cells undergoing neoplastic transformation by the oncogenic viruses will be discussed. Polyamine metabolism has been studied in several virus-infected cell systems. Among the systems most extensively studied in vitro are cell transformations by the DNA papovaviruses, such as polyoma or SV40, and by the RNA viruses, such as Rous sarcoma virus or murine sarcoma virus.

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A. EFFECTSO F ONCOGENIC RNA VIRUSES

RNA tumor viruses possess attractive features for study of the mech. anisms involved in cell transformation. These viruses cause rapid and highly efficient transformation which is reproducible under welldefined cellular conditions. The changes can be induced synchronously, so that sequential events can be analyzed. Systematic studies of transformation of chick embryo fibroblasts by different strains of Rous sarcoma virus were carried out by Bachrach and his co-workers. These authors found that transformed chick embryo fibroblasts did not differ from normal counterpart cells in their changes in protein and RNA content occurring during growth (Bachrach et al., 1973, 1974). However, transformation is accompanied by significant alterations in polyamine content, since a continuous rise in putrescine content was observed in transformed cells but not in normal ones, in which the intracellular content of this diamine reached a plateau when the cell became confluent (Bachrach et al., 1973, 1974; Bachrach, 1978).The difference between normal and transformed cells was magnified during subculturing or in old cultures (Bachrach et al., 1974). No significant differences between normal and transformed cells were noticed in the intracellular concentrations of spermine and spermidine (Bachrach et al., 1973, 1974). A connection of this increase in intracellular putrescine content with the viral transformation has been demonstrated with a temperature-sensitive mutant of Rous sarcoma virus, which induces transformation only at the permissive temperature (37°C) but multiplies at both the permissive and the nonpermissive (42°C) temperatures. The chick embryo fibroblasts increased their putrescine content only when viral infection by the temperaturesensitive mutant was carried out at permissive temperature (Don et al., 1975; Bachrach, 1978). When the temperature of transformed chick embryo fibroblasts was shifted from 37°C to 42"C, putrescine content markedly decreased, whereas putrescine content rose dramatically when the temperature shift was reversed, from 42°C to 37°C (Don and Bachrach, 1975; Don et al., 1975; Bachrach, 1978). The same was found to be true for in vivo infections of chorioallantoic membranes of chick embryos with the same strains of Rous sarcoma virus. Infection at the permissive temperature caused an increase in spermidine and putrescine content, and no significant change in the content of these two polyamines occurred between uninfected membranes and membranes infected at the nonpermissive temperature (Don et al., 1975). This makes it possible to distinguish between virus multiplication and the transformation process and to see if either is

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connected with some specific alterations in intracellular polyamine content. Further proof of this was provided by showing that chorioallantoic membranes infected with different types of nononcogenic viruses had the same polyamine levels as uninfected membranes (Don et al., 1975). ODC activity has been measured in the chick embryo fibroblasts using a temperature-sensitive mutant of Rous sarcoma virus and shifting of the temperature (Don and Bachrach, 1975; Bachrach, 1978). ODC activity has been shown to behave similarly and in parallel with that described above for intracellular putrescine content, i.e., it rose after viral transformation at the permissive temperature and dropped nearly to control levels when the temperature was shifted to the nonpermissive level (Don and Bachrach, 1975; Bachrach, 1978). It is worth noting that Rous sarcoma virus induced ODC enhancement before the appearance of morphological alterations in the cell (Don and Bachrach, 1975; Bachrach, 1978). Therefore, ODC elevation is also an early metabolic change in the cells related to viral transformation. Moreover, one of the effects of cell transformation with different strains of Rous sarcoma virus is a lengthening of the ODC half-life, which also occurred only at the permissive temperature (Bachrach, 1976a, 1978). Like ODC, the SAMD activity was increased by viral transformation under permissive conditions, whereas uninfected cells and those infected at the nonpermissive temperature had nearly the same low levels of enzyme activity (Bachrach and Wiener, 1980).Also like ODC, SAMD responded to temperature shifting from nonpermissive to permissive, but the increase in the enzyme activity was slower than that of ODC (Bachrach and Wiener, 1980). Unlike ODC, the half-life of SAMD did not significantly change in chick embryo fibroblasts transformed with the same temperature-sensitive mutant of Rous sarcoma virus at the permissive temperature (Bachrach and Wiener, 1980). The increase in ODC activity in virus-transformed cells has also been demonstrated with other cell types and with other RNA viruses. In fact, using mouse BALB/3T3 cells and murine sarcoma virus, which contains both a transforming virus and a nontransforming strain of murine leukemia virus acting as a “helper” virus, it was demonstrated that ODC activity markedly rose in cells acutely infected and transformed by murine sarcoma virus (Gazdar et al., 1976; Bachrach, 1978; Kilton and Gazdar, 1978). In contrast, infection with the nontransforming virus, the helper virus, had no effect on the cellular ODC levels (Gazdar et al., 1976; Bachrach, 1978; Kilton and Gazdar, 1978). Again, infection with Rauscher leukemia virus, another nontransforming virus, caused no enhancement of cellular ODC levels

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(Gazdaret al., 1976).The enhancement of cellular ODC activity seems truly to be connected with the acute transformation process, since ODC levels fell with successive cell passages, while different permanently transformed cell clones showed a wide range of ODC activities (Kilton and Gazdar, 1978). The cultured rat fibroblasts transformed by Rous sarcoma virus had a higher resting ODC level than the normal cells (Haddox and Russell, 1979). Like normal cell lines (see Section 111,C72),cultured fibroblasts transformed by either Rous sarcoma virus or temperature-sensitive mutant responded to the addition of either TPA or serum by enormously increasing ODC activity (Haddox et al., 1979,1980). However, the stimulation of ODC activity by serum, unlike that by TPA, was followed by increased intracellular content of polyamines in both normal and transformed cell lines tested (Haddox et al., 1979). Serum-dependent ODC induction in both normal and transformed cells was inhibited completely or nearly so by cycloheximide or actinomycin D (Haddox et al., 1980). The ODC increase in transformed cells in response to fresh media and serum addition was greater than in the normal cells (Haddox and Russell, 1979). Again, in cells infected with a thermosensitive mutant of Rous sarcoma virus, the induction of ODC activity after the addition of medium containing fresh serum was much greater at the permissive temperature than at the nonpermissive temperature (Haddox et al., 1980). It is of importance to note that ODC activity is induced to a greater extent during the G1 phase in viral-transformed fibroblasts than in the G1 phase of their normal counterparts (Haddox and Russell, 1979; Haddox et al., 1980), at which phase of the cell cycle the cells are well known to be more susceptible to malignant transformation by chemicals or by viruses (Baserga, 1977). This is notably due to the increased accessibility of chromatin in G1 cells to carcinogenic agents (Baserga, 1977). Furthermore, the enzyme increase in normal cells was totally dependent on serum growth factors, whereas in the Rous sarcoma virustransformed cell lines it was not, since addition of fresh medium alone to the transformed cells was enough to induce ODC activity (Haddox et al., 1980). Another difference between normal and transformed cells in ODC regulation is the lesser sensitivity of serum-dependent ODC induction to inhibition by putrescine in transformed cells than in normal ones (Haddox et al., 1980). All these results raise the possibility that altered regulation of ODC activity is one of the key features of the neoplastic transformation by viruses. Finally, polyamines can regulate the genome expression of mouse

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mammary tumor virus in mouse mammary tumor cells grown in semisynthetic medium (Svec and Links, 1977). In the absence of serum, the production of mouse mammary tumor virus was stimulated by spermidine, but not by spermine (Svec and Links, 1977). However, the stimulation by spermidine was by far less than that elicited by dexamethasone under the same experimental conditions (Svec and Links, 1977). In cultures preincubated with serum, no increase in the synthesis of mouse mammary tumor virus after spermidine treatment was observed (Svec and Links, 1977). B. EFFECTSOF ONCOGENICDNA VIRUSES Using mouse 3T3 cells transformed by SV40 virus, it was possible to show some differences between normal and transformed cells in the ODC responses to the same inducing stimuli (Lembach, 1974). First, untransformed cells required a significantly higher serum concentration to maintain comparable levels of ODC activity than did the transformed cells; this is in keeping with analogous results of Haddox et d. (1980) with a different experimental model. Second, marked increases in the level of ODC activity were observed in both untransformed and transformed cells when the extracellular concentration of serum was increased, but the increases in transformed cells were invariably higher than those in the normal ones (Lembach, 1974). This point was confirmed by Bethel1 and Pegg (1979a). Third, low serum levels that failed to induce ODC activity in 3T3 cells elicited increase in the enzyme level in transformed cells. Fourth, a serum free of gammaglobulins had little stimulating effect on ODC activity of normal cells, but in transformed cells significant increases in enzyme levels were elicited by that kind of serum (Lembach, 1974). Fifth, the SV40-3T3 cells required much greater concentrations of putrescine or spermidine to produce a decrease in ODC activity similar to that in their normal counterparts (Bethel1 and Pegg, 1979a,b). This suggests a possible difference between transformed and nontransformed cells in sensitivity to a decrease in ODC activity caused by the diamine or the polyamine. Also consistent with the idea that important qualitative differences is ODC regulation between normal and virus-transformed cells really exist in the significant finding that the specific activity of the ODC purified from an SV40-transformed 3T3 cell line is approximately eight times that of the ODC from normal tissues (Boucek and Lembach, 1977). In keeping with and in further support of the foregoing idea are the results of the investigations of Isom (1978, 1979). Arrested human fibroblast cells infected with human cytomegalovirus

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(which is a member of the herpesvirus group and is strongly suspected to be somehow involved in causing some human neoplasias) produced a stimulation of ODC activity far greater than that observed in mock infected cells (Isom, 1978, 1979). The ODC stimulation was specific for the viral infective process, since human cytomegalovirus inactivated by UV irradiation did not stimulate cellular ODC activity and human serum containing antibodies neutralizing the cytopathic effect of the virus in vitro also prevented the ODC stimulation (Isom, 1979). Moreover, by selectively blocking the activity of viral DNA polymerase without preventing the stimulation of cell DNA synthesis induced by the virus, ODC induction by virus was remarkably inhibited, suggesting that viral DNA synthesis is required for ODC induction (Isom, 1979). The significance of the abnormal ODC regulation in virus-infected cells is as follows: (1) The enzyme induction by virus was not sensitive to the inhibiting effect of putrescine added to growth medium, unlike the enzyme induction triggered by high serum medium in uninfected cells (Isom, 1978, 1979), but ODC prepared from either infected or uninfected cells was sensitive to the inhibitory action of polyamines in vitro (Isom, 1979; Isom and Backstrom, 1979). This insensitivity of ODC to putrescine was found to be peculiar to the late-passage cells (Isom and Backstrom, 1979). (2)In cells transformed by cytomegalovirus, the addition of fresh medium containing a high serum percentage enhanced ODC activity to a much greater extent than in normal cells (Isom and Backstrom, 1979). (3) ODC of the early-passage transformed cells was completely resistant to the inhibitory activity of spermidine, whereas ODC of the late-passage cells was less inhibited than that of the normal ones by this polyamine (Isom and Backstrom, 1979). Infection of mouse kidney cell cultures with polyoma virus caused biphasic increases in the activities of ODC and SAMD as well as in the intracellular levels of the three chief polyamines (Goldstein et al., 1976; Heby et al., 1976, 197813).This biphasic response consisted of a first peak that occurred shortly after the infection but before the onset of virus-induced host cell DNA synthesis and a second peak that occurred much later, corresponding temporally with the peak of virusinduced cell DNA synthesis (Goldstein et al., 1976; Heby et al., 1976, 1978b).With different types of inhibitors, it was possible to investigate better the connections between polyamine biosynthetic decarboxylases and DNA and rRNA syntheses. When 5-fluorodeoxyuridine completely blocked the virus-induced synthesis of cellular DNA, the time courses of the changes in ODC and SAMD activities and the polyamine levels in the infected cells treated with the inhibitor were

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virtually the same as those in the untreated cells (Goldstein et al., 1976; Heby et al., 1978b). Using actinomycin D at some critical values for its concentrations in the medium and the time of the exposure of the cells to the inhibitor increased ODC activity in virus-infected cells, i.e., ODC “superinduction” occurred (Goldstein et al., 1976; Heby et al., 1976, 1978b). SAMD activity was very seldom superinduced. Cycloheximide inhibited the activities of both the polyamine biosynthetic decarboxylases in the polyoma virus-infected cells (Goldstein et al., 1976; Heby et al., 1976, 197813). All these results show that in certain instances polyamine biosynthesis can be uncoupled from DNA and rRNA syntheses and that ODC and SAMD syntheses can be regulated independently. Polyamine uptake and metabolism have also been investigated in different cell lines transformed by different herpesviruses. The rate of putrescine uptake into MRC-5 cells was found to be increased markedly immediately after infection by human cytomegalovirus (Tyms and Williamson, 1980). The incorporated putrescine served as a precursor for spermidine and spermine syntheses, since the syntheses of these two polyamines were remarkably enhanced in the cells after viral infection and paralleled the putrescine changes (Tyms et al., 1979; Tyms and Williamson, 1980). Increase in the synthesis of virus DNA was also observed concomitantly with. the stimulation of polyamine metabolism (Tyms and Williamson, 1980).Conversely, the conversion of labeled ornithine or putrescine to labeled spermidine and spermine in different cell lines (both normal and neoplastic) infected with herpesvirus-1 or herpesvirus-2 was decreased (Gibson and Roizman, 1971, 1973; McCormick and Newton, 1975; McCormick, 1978b; Tyms et al., 1979), although an initial increase in the rate of putrescine uptake has been observed after cell infection with this kind of herpesvirus-1 (McCormick and Newton, 1975). These effects seem to be the result of inhibition of host protein synthesis after infection (McCormick and Newton, 1975). Unfortunately, the inhibition of polyamine metabolism in cultured cells infected by herpesvirus was not confirmed by Francke (1978), who found a large and steady increase in cell putrescine concentration and a small increase in spermidine concentration without changes in the spermine. There was also a difference in polyamine metabolism between normal and neoplastic cells, both infected by herpes simplex virus type 1 (HSV-l), in that mouse fibroblasts (L-cells) became extremely permeable to polyamines early in infection, whereas neoplastic cells (HeLa cells) did not (McCormick, 1978b).

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Finally, spermidine and, more especially, spennine, which is associated with the viral DNA in HSV-1 (Gibson and Roizman, 1971; Roizman and Furlong, 1974), have been shown to be positive effectors of the HSV-1 DNA polymerase (Wallace et al., 1980). In contrast, putrescine, which is not present in the virion, had little effect on the polymerase reaction (Wallace et al., 1980). Furthermore, the triamine and the tetraamine, but not the diamine, inhibited the deoxyribonuclease induced in KB cells by HSV-1 or HSV-2 ( H o h a n n and Cheng,

1978).

In conclusion, the qualitative differences in ODC regulation between normal and virus-transformed cells seem to us of particular interest as a promising direction to follow using different cell lines transformed by several oncogenic viruses. It is, in fact, well known that the viral transformation process of the cells causes alterations in the cell membrane and cell functions (Nicolau, 1978). It is therefore possible that changes in the receptor sites for putrescine and spermidine are responsible for the decreased sensitivity of the ODCs of some different virus-transformed cell lines to the inhibiting effects of the diamine and the polyamine. This concept is in keeping with the suggestion of Canellakis et al. (1978)that malignant cells continue to synthesize and excrete putrescine and polyamines even in the presence of extracellular polyamine concentrations that are known to normally depress ODC activity inside the cell. V. Changes in Polyamine Biosynthesis and Content of Target Tissues by Physical Carcinogens

Among the physical carcinogens, only the effects of ultraviolet light on polyamine biosynthesis in target tissue have been tested. The UV spectrum, the portion of the electromagnetic spectrum between visible light and X rays, is conventionally divided into three major regions: short-wave, with a wavelength of 250-290 nm (also called “germicidal” light), sunburn (UV-B), and long-wave, with a wavelength of 320-400 nm. Ultraviolet light is a complete carcinogen, possessing both initiating and promoting properties. The effective carcinogenic range of UV light is 250-320 nm (Freeman, 1975). ODC has been induced in the epidermis of several species of mammals by UV of different wavelengths. In hairless mice, very shortly after exposure to UV light (UV-B, mostly 290-320 nm), the epidermal ODC began to rise, reaching a peak at approximately the end of the

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first day after irradiation, and then declining gradually (Lowe et al., 1978; Boutwell et al., 1979; Verma et aZ., 1979b; Lowe and Breeding, 1980a,b; Peterson et al., 1980). ODC induction was found to increase progressively with increased numbers of exposures of mouse skin to ultraviolet light in the sunburn range (Lowe and Breeding, 1980b). The induction of epidermal ODC activity by UV-B irradiation was found to be dose dependent (Boutwell et al., 1979; Verma et al., 1979b). The magnitude of ODC induction also paralleled the histopathological lesions that could be observed (Verma et al., 1979b; Lowe and Breeding, 1980b). The time course of incorporation of tritiated thymidine into epidermal cells was not at all parallel to the time course of ODC in the same cells, since DNA synthesis decreased during the entire time that ODC activity increased and was enhanced when ODC declined (Lowe et aZ., 1978; Verma et al., 1979b). ODC induction in mouse epidermis by UV-B light appears to require de nooo synthesis of both protein and RNA, since injection of cycloheximide shortly before killing or 5-azocytidine shortly before UV-B irradiation greatly diminished the ODC stimulation (Verma et al., 1979b). SAMD activity was also induced in mouse epidermis after UV-B irradiation, but the increase was slightly delayed with respect to the ODC response (Boutwell et al., 1979; Verma et al., 197913). ODC induction by UV has also been observed in depilated human epidermis, but the maximum of the induction was later than in mice, occurring 48 hr after irradiation (Lowe et al., 1980). Germicidal ultraviolet light was shown to induce ODC activity in cultured mouse epidermal cells (Lichti et al., 1979, 1980) and to modify the polyamine pattern of mouse epidermis in vivo (Seiler and Knodgen, 1979a). The ODC induction was biphasic and was prevented by exposing the cells to actinomycin or cycloheximide, suggesting the involvement of both transcriptional and translational control of ODC induction (Lichti et al., 1979, 1980). ODC induction was dose dependent, within limits (Lichti et aE., 1980). When the cells were irradiated with germicidal ultraviolet light and then treated with TPA, the effects were additive only under certain particular experimental conditions, suggesting that induction of ODC by UV and its induction by TPA can be reasonably assumed to go through at least partially separate pathways (Lichti et al., 1980). The epidermal polyamine pattern was modified in vivo by germicidal ultraviolet light (Seiler and Knodgen, 1979a). Putrescine concentration rapidly increased within a few hours after irradiation and then declined gradu-

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ally. Spermidine concentration increased as well, but at a much slower rate than that of putrescine, and remained at elevated levels for a much longer time than putrescine. Spermine concentration decreased and, amazingly, reached its minimum when DNA synthesis was greatest (Seiler and Knodgen, 1979a). Whether ODC induction in epidermis by ultraviolet light is relevant to the carcinogenesis triggered by it is still only speculative. Part I of this review has covered polyamines and their metabolism in normal tissues and in chemical, physical, and viral carcinogenesis and cell transformation. Part I1 will appear in Volume 36 and will cover various aspects of polyamines in cancer; namely, polyamine biosynthesis and concentrations in different lines of cultured neoplastic cells; polyamines in human oncology; diamine oxidase activity in human or experimental neoplasms; physiological and pharmacological inhibitors of polyamine biosynthesis in neoplastic tissues or cells; and concluding remarks and speculations for both Parts I and 11.

Ac KNOWLEDGMENTS First, we are very grateful to Professor S. Weinhouse (Philadelphia), for his understanding of our delays. Thereafter we wish to thank those authors who kindly sent to us manuscripts of unpublished but accepted papers: Dr. E. S. Canellakis (New Haven), Dr. K. Y. Chen (New Brunswick), Dr. S. S. Cohen (Long Island), Dr. H. Desser (Vienna), Dr. J. M. Gaugas (Northwood), Dr. 0. Heby (Lund), Dr. U. Lichti (Bethesda), Dr. P. S. Mamont (Strasbourg), Dr. P. McCann (Cincinnati), Dr. D. Morris (Washington), Dr. K. Nishioka (Houston), Dr. G. Quash (Lyons), Dr. A. M. Roch (Lyons), Dr. N. Seiler (Strasbourg), and Dr. T. Slotkin (Durham). Although we have continued to reevaluate the topic while writing and have included new papers as they appeared, we know that we have not avoided all errors or lacunae. We apologize to those investigators whose works we have inadvertently not cited. We are deeply indebted to Professor E. Ciaranfi (Milano), who several years ago introduced us to this field and taught us to love polyamines. We also thank Professor A. Bemelli-Zazzera (Milano) for his interest and advice. We gratefully acknowledge the helpful criticism by Professor U. Bachrach (Jerusalem)of our outline for this work. One of us (G. S.) also thanks Professor J. Janne (Helsinki), in whose laboratory he had the opportunity several years ago to deepen his understanding of some modem aspects of polyamine biosynthesis regulation. To our young co-workers, Dr. M. Puerari and Dr. D. Modena, we express our gratitude for their patient help in organizing and revising the manuscript. Last, but not least, to Dr. B. Rubin (Milano) we extend our warmest thanks for her invaluable editorial assistance in revising the English of the manuscript.

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