Polyamines in Mammalian Tumors Part ii1

POLYAMINES IN MAMMALIAN TUMORS PART Ill Giuseppe Scalabrino and Maria E. Ferioli Institute of General Pathology and C N R Centre tor 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, u t 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 I’avons, par longue estude, confirmee e t averee. MONTAIGNE, “Essais,” L. 11, C. 12

I. Polyamine Biosynthesis and Concentrations in Different Lines of ........ Cultured Neoplastic Cells . . . A. Responses to Microenviron perature, PO,) and to the Presence of Different Exogenous Molecules (Amino Acids, Di- and Polyamines, Antipolyamine Antibodies) . . B. I n Relation to the Growth Rate and the Phase of the Cell Cycle . . . . . . . C . Two-way Relationships between Polyamines and Cyclic Nucleotides. Inducibility of the Two Polyamin D. Effects of Infection with Nononcogenic Vir E. Miscellaneous Effects of Polyamines . . . . . . 11. Polyamines in Human Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ A. Patterns of Polyamines in Human Neoplastic Tissues B. Levels of the Chief Polyamines a Urines of Normal Subjects and of C. Levels of the Chief Polyamines and Their Conjugated Forms in Blood, Plasma, Serum, Formed Blood Elements and Bone Marrow of Normal ................ Subjects and of Cancer Patients D. Levels of the Chief Polyamines ....................... Bloodand Urine . . . . . . . E. Levels of Activity of P Neoplastic Tissues in Relation to the Degree of Malignancy F. Metabolic Conjugation .............. Normals and in Cancer Patients 111. Diamine Oxidase Activity A. In Human Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. In Experimental Tumors ................

2 2 11 12 18 18 20 24

38

49 53 55 56 57 60

I Part I of this review (see Volume 35 of this series) covered polyamines and their metabolism in normal tissues and in chemical, physical, and viral carcinogenesis.

1 ADVANCES IN CANCER RESEARCH, VOL. 36

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any fonn reserved. ISBN 0-12-006636-X

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GIUSEPPE SCALABRINO A N D MARrA E. FERIOLI

IV. Physiological and Pharmacological Inhibitors of Polyamine Biosynthesis in Neoplastic Tissues or Cells ............................... A. Physiological Inhibitors and Related Compounds ..................... B. Pharmacological Inhibitors .......................................... V. Concluding Remarks and Speculations ................................... References .............................................................

62 63 68 86

88

I. Polyarnine Biosynthesis and Concentrations in Different Lines of Cultured Neoplastic Cells

A. RESPONSES TO MICROENVIRONMENTAL CONDITIONS (OSMOLARITY, TEMPERATURE,

p o z ) AND TO THE PRESENCE OF

DIFFERENT EXOGENOUS MOLECULES(AMINOACIDS,

POLYAMINES,

D I - AND

ANTIPOLYAMINE ANTIBODIES)

The need for expediency in experimental cancer studies has made the cultured neoplastic cell the principal tool for cancer research. There are obvious advantages in working with uniform cell lines that can be prepared as clean cell suspensions. However, the artificial conditions ofin vitro culture tend to change the characteristics of the cells. Several authors have studied different neoplastic cell lines growing in culture in order to delineate the metabolic pathways of the polyamines and to affect growth of these cells by selectively inhibiting polyamine synthesis or sequestering polyamines in order to define their roles (see also Section IV). It is particularly interesting to modify the culture conditions in order to clarify the influence of the environmental milieu on the activities of the polyamine biosynthetic enzymes by enhancing or decreasing the levels of these enzymes to see the role of polyamines in the cell growth process. To this purpose, several studies were carried out with rat hepatoma cells growing in culture. Studies of the effects of growth conditions on ornithine decarboxylase (ODC) activity presented evidence that dilution of high-density hepatoma cell cultures with fresh medium resulted in a very large and transient increase in enzyme activity, reaching a peak about 4 hr after dilution (Hogan, 1971; Hogan and Blackledge, 1972; Hogan e t d.,1973; Hogan and Murden, 1974). This increase was abolished by cycloheximide but not by actinomycin D, suggesting that ODC induction is controlled under these experimental conditions at the posttranscriptional level (Hogan, 1971; Hogan and Blackledge, 1972). The aforementioned increase of ODC activity appeared to be in part concomitant with a decrease in the rate of ODC degradation, i.e., with an increase in the half-life of ODC, hinting at a

POLYAMINES IN MAMMALIAN TUMOHS

3

causal relationship between the two phenomena (Hogan and Blackledge, 1972; Hogan et al., 1973; Hogan and Murden, 1974). The supplementation of high-density hepatoma cell cultures with glutamine or serum or nonessential amino acids, but not with essential amino acids, produced an ODC stimulation of severalfold, at least partially due to an increase of the half-life of ODC (Hogan et al., 1973, 1974; Hogan and Murden, 1974; Fong et ul., 1976). On the contrary, very high concentrations of pyridoxal do not affect the apparent halflife of ODC (Hogan and Murden, 1974). Other investigators followed ODC activity and putrescine levels through two generations of rat hepatoma cells cultured in the presence of serum (McCann et al., 1975). Biphasic ODC induction was noted during the first 24 hr. The intracellular putrescine concentration was found to correlate with rises in ODC activity (McCann et al., 1975). On the contrary, only one broad peak of ODC activity was observed over the same period in diluted hepatoma cell cultures without serum, with no parallel increase in the cellular putrescine content (McCann et al., 1975). Therefore, these authors concluded that only growing and dividing hepatoma cells have biphasic ODC induction that parallels increased putrescine levels, whereas a single peak of ODC stimulation can be achieved in nongrowing cells. Among the factors affecting the growth conditions of the cultured cells, the addition of fresh medium or serum to the culture has been demonstrated to be one of the most important for a variety of other cell lines. Induction of ODC activity, followed by a significant elevation of cellular putrescine concentration, in a rat glioma clone and in a mouse neuroblastoma cell clone when fresh medium was added to confluent cultures was reported (Bachrach, 1976c, 1977, 1980a; Bachrach e t al., 1978). In more detail, ODC activation following the addition of fresh serum was preceded by similar responses in both adenosine 3’ : 5’cyclic monophosphate (cyclic AMP, CAMP)-dependent and CAMPindependent protein kinases of glioma cells (Bachrach et al., 1978). However, in this case no difference in the half-life of ODC before and after the addition of fresh medium was observed (Bachrach, 1 9 7 6 ~ ) . Moreover, the induction of ODC activity appears to be specific for this enzyme, since the activities of other enzymes that decarboxylate other amino acids were not stimulated by the addition of fresh medium (Bachrach, 1 9 7 6 ~ ) This . study also suggests a correlation between growth rate and ODC activity in cultured glioma and neuroblastoma cells, since the enzyme activity was high when the cells were proliferating rapidly (Bachrach, 1 9 7 6 ~ )Interestingly . enough, the addition of serum to culture medium containing mouse neuroblastoma cells or

4

GIUSEPPE S C A L A B R I N O A N D MARIA E. FERIOLI

rat glioma cells also greatly increased formation of y-aminobutyric acid (GABA) from putrescine (Kremzner et al., 1975; Sobue and Nakajima, 1977). The S-adenosyl-L-methionine decarboxylase (SAMD) activity was also increased in glioma and neuroblastoma cells shortly after the addition of complete fresh medium (Bachrach, 1977, 1980a). As expected, the enhancements of the activities of the polyamine biosynthetic decarboxylases were found to be paralleled by increases in cellular concentrations of polyamines and of y-aminobutyric acid formed from putrescine (Bachrach, 1980a). In HeLa cells, in response to the addition of serum to quiescent cells not only ODC activity increased but also SAMD activity (Prouty, 197613; Maudsley et al., 1978). Under the same experimental conditions, putrescine and spermidine levels markedly increased as well (Maudsley et al., 1978). When labeled ornithine was added to the cells during the period of the serum stimulation and its uptake was measured, a marked and rapid increase in polyamine levels above that normally observed in resting cells was noted (Maudsley et al., 1978). It appeared that most of the cytosol ornithine was decarboxylated to yield putrescine, which in turn was quickly converted to spermidine (Maudsley et al., 1978). More or less analogous observations were made in KB cells (Pett and Ginsberg, 1968) and in hepatoma cells (Bondy and Canellakis, 1980). In HeLa cells growing in suspension culture, ODC activity was also found to be potently stimulated by the addition of glutamine to the medium; this stimulation was due, at least partly, to a decrease in the rate of decay of the ODC activity (Prouty, 197613). In cultures of L1210 mouse leukemic cells, of hepatoma H35 cells, of neuroblastoma cells (Chen et al., 1976a,b), of virally induced glioma-like hamster brain tumor cells (Hsu et al., 1977), and of Friend erythroleukemia cells (Tsiftsoglou and Kiriakidis, 1979; Gazitt and Friend, 1980), the addition of fresh medium plus serum also resulted in an increase in ODC activity. Additionally, when Friend leukemia cell cultures were stimulated to proliferate by dilution of stationaryphase cultures with fresh medium, both their nucleolar RNA synthesis rates and ODC levels were increased (Dehlinger and Litt, 1978).The addition of putrescine at the time of dilution with fresh medium blocked the increase in ODC levels, but did not prevent the increase in nucleolar RNA synthesis (Dehlinger and Litt, 1978).As observed by Ferioli et al. (1980) in postischemic liver repair, ODC induction in Friend leukemia cells can be dissociated from the stimulation of RNA synthesis.

POLYAMINES IN MAMMALIAN TUMORS

5

A proliferative stimulus for the cultured cells, such as the addition of serum, was followed by a dramatic increase in the rate of putrescine transport into both normal human fetal lung fibroblasts and the same cell line transformed by SV40 (Pohjanpelto, 1976). Conversely, the removal of serum resulted in a rapid decrease in the rate of putrescine transport. The magnitude of the increases or of the decreases in the rates of putrescine transport in these two cell lines in response to the addition or the removal of serum were nearly the same (Pohjanpelto, 1976). For studies of the effects of the addition of fresh serum, 12-0tetradecanoylphorbol-13-acetate(TPA), and/or a combination of the two on ODC activity of cultured malignant cells, the reader is referred to Section III,C,2,b, Part I, Vol. 35. Besides the previously mentioned glutamine, another a-amino acid with nonionic polar side chains, asparagine, is a powerful inducer of ODC activity in confluent neuroblastoma cells (Chen and Canellakis, 1977). Among the natural amino acids tested, asparagine led in ability to induce ODC, with L-glutamine second, half as effective as asparagine (Chen and Canellakis, 1977). This ODC induction was neither concomitant with nor followed by an increased rate of incorporation of precursors into DNA, RNA, or proteins (Chen and Canellakis, 1977). What is really astonishing is the finding that asparagine and glutamine play a “permissive” role in ODC induction by N 6 , 0 2 ’ dibutyryl cAMP or by prostaglandin E l (PGEl) plus S-isobutyl1-methylxanthine, since none of these three molecules alone stimulated ODC activity at all in confluent neuroblastoma cells in a medium devoid of asparagine or glutamine (Chen and Canellakis, 1977). The “stabilizing” effect of asparagine on ODC was demonstrated b y the very great lengthening of the enzyme’s half-life when asparagine was in the medium (Chen and Canellakis, 1977). Moreover, in mouse neuroblastoma cells induced to differentiate by any of several stimuli, the regulation of ODC induction by asparagine in undifferentiated and in differentiated cells was studied comparatively (Chen, 1979, 1980). The addition of asparagine to a salts-glucose medium elicited a maximal increase of ODC activity in undifferentiated cells with further addition of fetal calf serum or of N6,02’-dibutyryl cAMP not resulting in any additional increase (Chen, 1979, 1980). In contrast, the addition of asparagine alone caused a small increase in ODC activity in differentiated cells, and this increase was potentiated and reached a maximum after addition of fetal calf serum or of N 6 , 0 2 ’ dibutyryl cAMP (Chen, 1979, 1980).

6

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

In the presence of asparagine, confluent glioma cells exhibited an increase in ODC activity whereas the SAMD activity of the same cells under the same experimental conditions remained at the basal levels (Bachrach, 1980a). That asparagine stabilizes ODC, leading to a lengthening of the enzyme half-life and to an apparent increase in its activity, was confirmed (Bachrach, 1980a). These results are in good agreement with those of Chen and Canellakis (1977). Furthermore, the extents of ODC induction by asparagine were compared in normal and in transformed fibroblasts (Costa, 1979; Costa and Nye, 1979). Enhancement of ODC by this amino acid was much greater in cells transformed either by SV40 or by Ni& than in the counterpart normal cells (Costa, 1979; Costa and Nye, 1979). Despite the numerous investigations, the exact role of asparagine in influencing cellular polyamine metabolism remains to be elucidated, and further studies are needed to achieve a better understanding of the mechanisms by which this amino acid specifically affects ODC activity inside the cells. However, among the various inducers of ODC activity in cultured cells mentioned so far, GABA has been shown to be remarkably more effective in enhancing ODC activity, at least in cultured rat hepatoma cells, than the amino acids asparagine and glutamine (McCann et al., 197913). Like asparagine, GABA seems to have a direct stabilizing effect on ODC with a consequent slowing down of the enzyme’s turnover and a concomitant lengthening of its half-life (McCann et al., 1979b). The cellular putrescine levels after addition of GABA to the culture medium increased in parallel with the increases in ODC activity (McCann et al., 1979b). However, addition of GABA modified neither the cellular spermidine and spermine concentrations nor the SAMD activity of the tumor cells (McCann et al., 197913). Whether or not GABA has a general role in the comprehensive complex regulation of ODC activity in eukaryotic cells or, on the contrary, has only a limited role in particularly specialized cells, such as brain cells, with elevated GABA concentrations, remains a matter for speculation. Another environmental factor influencing basal ODC activity and polyamine contents in cultured cells is the osmolality of the surrounding medium. In HeLa cells the polyamine contents were found to be inversely related to the osmolality of the growth medium (Munro et al., 1975). A sudden increase in NaCl concentration of the medium causes a rapid fall in putrescine and spermidine concentrations. A sudden decrease in NaCl in the medium causes a rapid increase in putrescine (Munro et al., 1975). The levels of ODC activity in relation to external osmolality behaved like the polyamine contents and were, therefore, consistent with the changes in the polyamine levels (Munro

POLYAMINES IN MAMMALIAN TUMOHS

7

et al., 1975). The ODC soon declined when the external NaCl concentration rose and increased when the osmolality decreased. Interestingly enough, these variations in ODC activity were accompanied by similar variations in the half-life of the enzyme, since the half-life decreased when the enzyme activity decreased and increased when the enzyme activity increased (Munro et al., 1975). Nevertheless, suitable concentrations of intracellular cations are also important for regulating ODC activity, at least in L1210 mouse leukemic cells. It has been demonstrated that several ionophore antibiotics (which are compounds produced by microorganisms that specifically increase the permeability of the cell membrane to ions), such as valinomycin, nigericin, and monensin (valinomycin belongs to the group of the neutral ionophore carriers, and nigericin and monensin are carboxylic carriers) have the ability to strongly inhibit ODC activity, with only a slight decrease in protein synthesis (Chen and Kyriakides, 1977). The extracellular cations, in addition to regulating basal intracellular ODC activity, play essential roles in influencing ODC induction. Accordingly, the addition of suitable amounts of MgClz or NaCl or KCI completely or nearly inhibited the rises in ODC activity induced in cultured H35 or neuroblastoma cells by the addition of fresh medium with or without a serum supplement (Chen et al., 1976b). These cations, when present in the induction medium, prevented ODC enhancement (Chen et d.,197613). And, what is of more interest, if the L1210 cells have been grown for several generations in a medium containing a high Mg2+ concentration, the ODC induction produced by diluting these cells during the stationary phase with a medium of high Mg2+concentrations, i.e., with a medium theoretically inhibiting the ODC response, surprisingly reached the same levels as in the controls (Chen et al., 197613). This means that the leukemic cells are able to adapt progressively to new environmental conditions, at least in ODC induction. Furthermore, these cations did not significantly modify macromolecular synthesis when they were present in the medium at the same concentrations at which they prevented ODC induction by serum or fresh medium (Chen et al., 197613). Hitherto we have considered mainly those external factors able to induce ODC activity inside cells in culture. There are other factors that can cause the converse effects. The exposure of cultured hepatoma, W256 carcinosarcoma, leukemic, or neuroblastoma cells to different substances, chiefly to putrescine added to the culture medium, greatly decreased the ODC activity inside the cells (Clark and Fuller, 1976; Fong et al., 1976; Heller et al., 1976a,b, 1977a, 1978;

8

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

McCann et al., 1977a; Heller and Canellakis, 1980). As discussed in detail in Section I,B,l, Part I, Vol. 35, under such experimental conditions putrescine elicits the synthesis of the ODC antizyme. Among the neoplastic cell lines tested, the only negative reports in this respect are those of Clark and Fuller (1976), who did not detect this ODC inhibitor in cultures of polyoma-transformed 3T3 cells after exposure to serum and putrescine, and of Kudlow et al. (1980),who did not note the presence of the soluble inhibitor of ODC activity in cultured cells of a mouse adrenocortical tumor, but without any pretreatment whatever with polyamine. In addition to putrescine, spermidine, spermine, cadaverine, and other unphysiological polyamines can elicit the synthesis of ODC antizyme in a great variety of cultured cell lines, both neoplastic and normal (Heller et al., 1977a, 1978; McCann et al., 1977a). It must be recalled here once again that the concentrations of di- or polyamines in the medium that are required to stimulate the ODC antizyme are several orders of magnitude smaller than the amounts of di- and polyamines present inside cultured cells (Canellakis et al., 1978; Heller et al., 1978). The half-life of the ODC antizyme in various cultured neoplastic cell lines has been shown to vary roughly proportionally with the variations of the half-life of ODC measured under the same experimental conditions (Heller et al., 1976b). Last, it should also be noted that ODC antizyme is normally present in cultured hepatoma cells, not stimulated for synthesis of this ODC inhibitor (Heller et al., 1977b). Under these resting conditions the antizyme exists as an inactive protein bound to subcellular components from which it can be liberated b y treatment with very low concentrations of polyamines, noticeably of putrescine, at concentrations far less than those usually found inside the cells (Heller et al., 197713). The use of heat alone for treatment of cancer patients dates back to the late nineteenth century. However, there has been renewed interest, and considerable emphasis has been placed on using this old treatment for cancer patients, i.e., hyperthermia, either alone or in combination with other types of antineoplastic therapy, usually with radiation (Manning, 1979). It is now widely known that virtually every fundamental phase of cellular biochemistry (respiration, glycolysis, DNA, RNA, and protein synthesis) can be disrupted by sufficient exposure of mammalian cells to hyperthermia, resulting first in a loss of proliferative capacity and ultimately in cell death. In spite of this, the molecular mechanisms by which hyperthermia kills cells or causes prolonged cell cycles are still not fully understood. Nonetheless, some interesting studies have dealt with this topic, elucidating some aspects of the problem. In synchronous Chinese hamster ovary cultures, pro-

POLYAMINES IN MAMMALIAN TUMORS

9

gressing through the cell cycle after exposure to 43°C for 1 hr during either the GI or the S phase, there is a remarkable leakage of polyamines, mainly spermidine and spermine, into the culture medium (Gerner and Russell, 1977; Gerner et al., 1980). Obviously, this has as natural consequence the depletion of intracellular spermidine and spermine (Gerner et al., 1980);this depletion was reversed when the temperature was reset at 37°C (Gerner and Russell, 1977). In contrast, the intracellular putrescine concentration was not affected b y exposure of the cells to heat shock (Gerner and Russell, 1977). It is reasonable to connect the depletions of intracellular spermidine and spermine, most probably due to membrane damage by heat, with the alterations in DNA synthesis observed in the same cell line under the same experimental conditions (Gerner and Russell, 1977). Again, polyamines have the property of potentiating the killing of the cells by heat. In fact, exposure of cultured Chinese hamster cells to hyperthermia plus a polyamine (cadaverine or putrescine or spermidine or spermine) in the growth medium resulted in dramatic, synergistic cell death, regardless of the order of the two treatments (Ben-Hur et al., 1978; Gerner et al., 1980). Spermine was the most effective polyamine for potentiating thermal cell killing, followed by spermidine, cadaverine, and putrescine, in order of effectiveness (Ben-Hur et al., 1978). When there was a long time interval between the two treatments, this synergism disappeared (Ben-Hur et al., 1978). This enhancement of thermal killing by polyamines is dependent on the time of exposure and on the concentration of the exogenous polyamines (Gerner et al., 1980).The minimal polyamine concentrations that enhance the thermal sensitivity of the cells were by far lower than those normally found intracellularly, strongly suggesting a membrane effect (Gerner et al., 1980). Moreover, prolonged hyperthermia caused an increase in the uptake of exogenous polyamines (with the exception of putrescine) added to the growth medium by the same cultured cell line (Ben-Hur and Riklis, 1978). Generally speaking, the polyamines that penetrated into the cells were metabolized into the same products at both the physiological and the high temperature, indicating that the enhancement by polyamines of cellular sensitivity to heat shock is due to these molecules as such, not to their metabolites (Ben-Hur and Riklis, 1978). Another line of evidence that the potentiation of hyperthermia-induced cytotoxicity by polyamines is specific for these polycations is that the effect was not obtained with mono- or divalent inorganic cations, such as KC1, CaCI2, and MgC12 at equimolar concentrations (Gerner et al., 1980). The importance of all the foregoing observations lies in the fact that neoplastic cells usually contain

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

larger amounts of polyamines, and this may be one reason for the wellknown higher sensitivity of cancer cells to heat. Hyperthermia also produced in cultured Chinese hamster fibroblasts a drastic decrease in ODC activity, which occurred rapidly and exponentially as a function of continued exposure to heat (Fuller et al., 1977; Ben-Hur and Riklis, 1979a; Gerner et al., 1980).When the temperature reverted to the physiological level, ODC activity recovered and returned to control levels, after overshooting (Ben-Hur and Riklis, 1979a). The activity of SAMD was affected in the same way as that of ODC b y hyperthermia (Fuller et al., 1977). Polyamines have been shown to potentiate the killing effect of heat on mammalian cells, and they can also amplify the synergism between heat and radiation. In fact, spermine enhanced the synergistic interaction between hyperthermia and y-radiation in cultured Chinese hamster fibroblasts (Ben-Hur and Riklis, 1979b).This property of spermine to further strengthen the radiosensitizing effect of heat on the cells resulted in (a) enhanced cell death in the presence of radiation plus heat plus tetraamine, as compared to radiation plus heat; and (b) a drastic inhibition of cell repair of radiation-induced sublethal damage (Ben-Hur and Riklis, 1979b). Unfortunately, the effects of exogenous polyamines on .cultured neoplastic cells exposed to hyperthermia have received little, if any, attention so far, and almost nothing is known about this topic. In addition to hyperthermia, severe hypoxia induces an arrest of the cell cycle, so that it has been, and might presently be, considered a potentially useful tool for cancer therapy. In this regard, there is an interesting report that Chinese hamster cells exposed to severe and prolonged hypoxia and then reoxygenated slowly reenter the cell cycle and progressively increase their protein and DNA syntheses, but, astoundingly, the ODC activity fails to increase when oxygen is supplied (Kehe and Harris, 1978). In this instance, as in hyperthermia, the effect of hypoxia on polyamine biosynthesis and metabolism in cultured neoplastic cells has never been tested. Cellular ODC activity ca'n be greatly inhibited by some mitotic poisons, such as colchicine and vinblastine, that disrupt the cellular microtubule system. Indeed, when these two drugs were added to the medium of cultured L1210 cells, the activation of ODC activity by the dilution of the cells was prevented (Chen et al., 1976a). In contrast, lumicolchicine, an isomer of colchicine without any effect on the microtubular system, did not inhibit the ODC rise under the same experimental conditions (Chen et a1., 1976a). Vinblastine and colchicine also blocked O D C induction in rat glioma cells by dibutyryl CAMP

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POLYAMINES IN MA.MMALIAN TUMORS

(Gibbs et al., 1979, 1980). In this cell line, too, lumicolchicine had no effect on either basal or stimulated levels of ODC activity (Gibbs et al., 1979, 1980). Furthermore, the integrity of the cytoskeleton seems to b e of great importance for ODC stimulation by added serum, since cytochalasin B inhibited ODC induction in L1210 cells (Chen et al., 1976a; Gibbs et al., 1979, 1980). Last, using an immunological approach, Quash and his collaborators (1971, 1972, 1973, 1978) have demonstrated that antipolyamine antibodies are cytotoxic for baby hamster kidney cells transformed b y the polyoma virus and growing in cell culture, and that complement is a necessary factor for the cytolytic effect, indicating the involvement of the cell membrane in the phenomenon. Moreover, cytolysis was inhibited and the cells recovered if the antipolyamine antibodies were removed or if putrescine, but not spermidine or spermine, was added to the culture medium containing the antiserum. Finally, evidence was provided that cytolysis of BHK-transformed cells is caused by the interaction of antidiamine antibodies with putrescine-containing sites on the cell membrane. This stresses once again the importance of the cell membrane in regulating ODC activity inside the cell.

B.

I N RELATIONTO CELL CYCLE

THE

GROWTHRATE

AND THE

PHASE OF

THE

Cultured cells of experimental neural neoplasias have been widely employed to investigate the connections between polyamine contents, combined or not with enzyme levels, and the growth rate of the cells. The activities of ODC and SAMD in a rat brain tumor cell line reached their maximum levels during the exponential growth phase and decreased as the growth curve reached a plateau (Heby et al., 1975b). The correlation coefficients obtained for the relationship of the enzyme activities to the specific growth rates were highly statistically significant (Hebyet al., 1975b).In studies of the correlations between cellular polyamine levels and the specific growth rate of the tumor cells, putrescine and spermine were not correlated, whereas spermidine and the spermidine : spermine ratio showed a direct positive linear correlation (Heby et al., 1975a,b). Parenthetically, it must be stressed that the rate of cell multiplication was maximal when the spermine content 1975b). Last, the compartmentalization of the was lowest (Heby et d., polyamines between nucleus and cytoplasm in this brain tumor cell line strongly indicates that spermidine and spermine act at the nuclear level, because the concentrations of these two polyamines were much

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GIUSEPPE SCALARRINO AND MARIA E . FERIOLI

higher in the nucleus than in the cytoplasm (Heby, 1977).There were no significant changes in putrescine levels between the two cellular compartments, although the ODC activity was located mainly in the cytoplasm (Heby, 1977). In mouse neuroblastoma cells and in rat glioma cells, the spermidine : spermine ratio was found to decrease when growth was less rapid, and the putrescine content decreased as the cells entered the stationary phase (Kremzner, 1973; Kremzner et al., 1975; Sobue and Nakajima, 1977). However, the metabolism of the polyamines in these two kinds of neural neoplastic cell lines was found to be different, since in neuroblastoma cells the formation of GABA from putrescine was low during the logarithmic phase of cell growth and increased astoundingly during the stationary phase, whereas in glioma cells this metabolic conversion was always low throughout both phases (Sobue and Nakajima, 1977). There seems to be an inverse correlation between the rate of polyamine biosynthesis and the size of the polyamine pool in HeLa cells. The contents of polyamines were the highest during mitosis and the late GI phase, while at these times polyamine biosynthesis was minimal (Sunkara et al., 1979~). On the other hand, the polyamine contents were the lowest during early the GI and S phases, while the polyamine biosynthesis was maximal (Sunkara et al., 1979~).However, conclusions drawn from studies carried out with synchronized cell populations have to be drawn with caution, since it has been demonstrated in HeLa cells that the synchronization protocols, which yield large numbers of synchronized cells, can deeply affect both the basal cell content of polyamines and the polyamine accumulation during the cell cycle (Goyns, 1980). Polyamine biosyntheses and their levels in normal cultured cells in the different phases of the cell cycle have been reviewed briefly by Pardee et al. (1978)and exhaustively by Heby and Anderson (1980).

c. TWO-WAYRELATIONSHIPSBETWEEN POLYAMINES AND CYCLIC NUCLEOTIDES. INDUCIBILITY OF THE TWO POLYAMINE BIOSYNTHETIC DECARBOXYLASES

As mentioned and discussed in Section I,E,l, Part I, Vol. 35, there are accumulated lines of evidence for CAMP as mediator of ODC induction in both in vitro and in vivo systems, with some arguments against it. We will now describe the experiments connected with this aspect carried out in cultured cells plus those experiments emphasizing the reverse aspect of the problem, i.e., the effects of polyamines on

POLYAMINES IN MAMMALIAN TUMORS

13

biosynthesis and metabolism of the cyclic necleotides in some cultured cell lines. ODC activity has been induced by cAMP and by dexamethasone (which is thought to act without implicating cAMP as second messenger) in logarithmically growing hepatoma cells originated from a Morris rat hepatoma and maintained in suspension culture (Canellakis and Theoharides, 1976).The induced enzyme was characterized by its immunoprecipitation and heat-stability patterns and shown to be identical with the enzyme purified from untreated hepatoma cells (Canellakis and Theoharides, 1976). ODC induction by these two drugs is similar in that the rates of ODC synthesis are markedly enhanced in both responses over that in controls (Canellakis and Theoharides, 1976). However, these two types of ODC induction have been shown to differ from each other in several aspects, namely, in their time courses and in their responsiveness to different types of inhibitors, such as actinomycin D and polyamine (Canellakis and Theoharides, 1976). First, the time course of ODC induction after dexamethasone was much slower than that after CAMP, but the enzymic levels were steadily elevated for many more hours. Second, actinomycin D completely inhibited induction by the glucocorticoid but only partially inhibited induction by CAMP.Third, the reverse is true for the effects of spermine and spermidine, since either these two polyamines depressed the ODC levels in the presence of cAMP even below basal control levels, whereas they were without effect on dexamethasone induction (Theoharides and Canellakis, 1975; Canellakis and Theoharides, 1976). Therefore the control of ODC activity in cultured hepatoma cells implies two paths, one CAMP-dependent and one CAMP-independent. All these results are in substantial agreement with those obtained with the same two drugs on another cell line of cultured hepatoma, i.e., the Reuber H35, by Byus et al. (1976) at the same time. ODC activity was induced in the H35 cells not only by dexamethasone and CAMP, but also by a series of 8-substituted cAMP analogs (Byus et al., 1976). Addition of insulin to H35 cultured cells was not followed by any increase in enzyme activity (Byus et al., 1976). Furthermore, the inducibility of ODC activity in H35 hepatoma cells has also been shown to be dependent on the composition of the culture medium (Liu and Chen, 1979). In vitro incubation of slices of rat adrenocortical carcinoma in the presence of cAMP resulted in significant ODC induction (Richman et al., 1973). More recently, a genetic approach has been used to explore whether hormonal activation of ODC activity is mediated by cAMP

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

and CAMP-dependent protein kinase (Kudlow et al., 1978, 1980). These authors used a cell line of a mouse adrenocortical tumor and its mutant clones, defective in either the adenylate cyclase response to ACTH or the response of the CAMP-dependent protein kinase to the same hormone. Both ACTH and cAMP induced ODC activity in the intact cell line, whereas only cAMP was able to enhance the enzyme levels in the mutant clones defective in adenylate cyclase (Kudlow et al., 1978, 1980). In the mutant clones defective in CAMP-dependent protein kinase, the magnitude of ODC response to ACTH was greatly reduced, but not totally abolished in some instances (Kudlow et al., 1978,1980). Both cAMP and nerve growth factor (NGF) induced ODC activity in a clonal cell line that originated from a rat adrenal pheochromocytoma, although the two types of induction were shown to be not causally interdependent, since NGF added to the culture medium did not produce any significant increase in cellular cAMP levels, even in the presence of theophylline (Hatanaka et al., 1978).The addition of either insulin or epidermal growth factor (EGF) (which both share some structural analogies with NGF) or of N2,02’-dibutyrylcGMP caused only little or no stimulation of ODC activity in this type of cultured neoplastic cell (Hatanaka et al., 1978). A concurrent report confirmed that ODC activity can be induced in these pheochromocytoma cells b y NGF and that the phenomenon requires new protein synthesis (Greene and McGuire, 1978). Furthermore, it was demonstrated that other noteworthy biological effects of NGF in these responsive cells, i.e., the stimulation of cell survival and of neurite outgrowth, were not impeded by total suppression of the cellular ODC activity by treatment with 1,3-diaminopropane or 5-hexyne1,4-diamine (Greene and McGuire, 1978). In contrast with the earliest report of Hatanaka et al. (1978), it was shown that E G F induced ODC in the rat pheochromocytoma clone PC12 and that preincubation of these cells in the presence of NGF largely prevented the ODC response to addition of E G F (Huff and Guroff, 1979). Both E G F and insulin stimulated putrescine transport into KB cells, but only insulin significantly enhanced the ODC levels of this type of cultured neoplastic cell (Di Pasquale et al., 1978). Cultures of tumors of the central nervous system are a good experimental model for clarifying the links between cyclic nucleotides and polyamine biosynthetic decarboxylases. The ODC activity of a rat glioma clone was quickly stimulated by addition of norepinephrine or isoproterenol or 3-isobutyl-1-methylxanthine (IBMX) or dibutyryl cAMP (Bachrach, 1975). In a mouse neuroblastoma clone, ODC activity was induced by PGE, or adenosine, but preincubation with IBMX

POLYAMINES I N MAMMALIAN TUMORS

15

was absolutely necessary to obtain the stimulatory effect (Bachrach, 1975).Complementary to this observation is another provided by Bachrach and his co-workers (1979), which demonstrates that morphine (an opiate that inhibits adenylate cyclase activity in some neural cell lines) almost completely antagonized the stimulating effects of IBMX with or without PGEl on the ODC activity and of the combination of the two drugs on CAMP levels in neuroblastoma x glioma hybrid cells (Bachrach e t al., 1979). In this case morphine also inhibited the stimulation of the activity of CAMP-dependent protein kinase elicited b y PGEl plus IBMX in the same cell hybrids (Bachrach et al., 1979). The assumption that CAMP is involved in SAMD induction too, was made and verified in the same cultured neoplastic lines (Bachrach, 1977). In fact, the level of this second polyamine biosynthetic decarboxylase was eleveted in glioma cells by the phosphodiesterase inhibitor IBMX or by catecholamines, and in neuroblastoma cells by PGE, and IBMX (Bachrach, 1977). All the foregoing results strongly support the idea that inductions of both the polyamine biosynthetic decarboxylases in cell cultures derived from neoplasms of the central nervous system of the rat or the mouse are mediated by CAMP and probably imply a wide cascade of biochemical events. However, Chen and Canellakis (1977) demonstrated that the stimulation of ODC activity in cultured mouse neuroblastoma cells brought about by the addition of N6,0Z’-dibutyrylcAMP or by PGE, plus IBMX was completely dependent on the presence of optimal concentrations of asparagine in the medium. Even more interesting, these authors also demonstrated that ODC could be induced in this cell line also b y high concentrations of asparagir,,. without any CAMP (Chen and Canellakis, 1977). An attempt to reconcile these seemingly contradictory reports of the true role of cAMP in ODC induction in neoplastic neural cell lines has been made by Gibbs et al. (1979,1980),who showed that there are separate pathways of ODC induction in rat glioma cells, i.e., one involving CAMPmediation and one not, and that these two pathways have as a common biochemical feature an absolute requirement for Ca’+. In fact, both isoproterenol and dibutyryl CAMP induced ODC activity in these neural cells, but both these inductions were completely abolished by the presence in the culture medium of ethylene glycol bis(p-aminoethyl ether)-N,N,N N ’-tetraacetic acid (EGTA), a well-known and powerful Ca2+chelator (Gibbs e t al., 1979, 1980); EGTA alone reduced the basal level of ODC activity (Gibbs et d., 1979, 1980). On the other hand, EGTA was also able to prevent ODC induction in the same cellular line by fetal calf serum, which last had little, if any, effect upon intracellular CAMP content (Gibbs et al., I,

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

1979, 1980). The addition of calcium alone without serum did not modify ODC activity to any appreciable extent, and the specifity of Ca2+requirement for ODC induction has been demonstrated by the fact that this ion cannot be substituted for magnesium (Gibbs et al., 1979, 1980).

The hypothesis that cAMP mediates the increase in ODC activity has also been tested in mouse S49 lymphoma cells (Insel and Fenno, 1977, 1978), with results opposite to those hitherto described. Incubation of cells of the wild-type of this lymphoma with dibutyryl CAMP, after an initial fleeting increase, profoundly and progressively decreased both ODC and SAMD activities to barely detectable levels (Insel and Fenno, 1977, 1978). Inhibition of ODC activity in S49 lymphoma cells is an early response to other agents that increase intracellular cAMP level, i.e., isoprotereno1, cholera toxin, or PGEl (Honeysett and Insel, 1980). These decreases in cellular ODC and SAMD levels were causally connected with a parallel decrease in the activity of CAMP-dependent protein kinase, since in “kinasenegative” mutant cells, that is, in a cell line totally lacking in CAMPdependent protein kinase activity and therefore in response to dibutyryl CAMP, no decrease in the levels of the two polyamine biosynthetic decarboxylases was observed after addition of this cyclic nucleotide to the culture medium (Insel and Fenno, 1977, 1978). On the contrary, the fall in ODC activity induced in the wild-type S49 cells by cAMP was not accompanied by a progressive decrease in protein synthesis, demonstrating that the two biochemical events are dissociable (Insel and Fenno, 1977, 1978). An analogous split between the decrease in the activities of the polyamine biosynthetic decarboxylases and the cell killing induced by cAMP is also possible, using the “CAMP-deathless” mutants, that is, mutants phenotypically resistant to the cytolysis induced by cAMP (Insel and Fenno, 1977, 1978; Kaiser et al., 1979). Furthermore, treatment with dibutyryl cAMP caused similar decreases in ODC activity in the ‘‘CAMP-deathless” mutants, regardless of the phase of the cell cycle at which the distinct cell populations were examined (Kaiser et al., 1979). Instead, Bachrach (1980b) found that ODC activity was induced by cAMP in cultures of S49 lymphoma cells, but not in a mutant line defective phenotypically in CAMP-dependent protein kinase activity. The aspect, which is converse and complementary to that hitherto analyzed, of the connections between polyamine biosynthesis and cyclic nucleotides, i.e., whether polyamines can modulate the metabolism of the cyclic nucleotides inside the eukaryotic cells, has been scarcely investigated. In spite of this, some evidence is emerging that pol yamines really can regulate the synthesis of the different cyclic

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nucleotides. Indeed, spermine dramatically inhibited the activity of CAMP-dependent protein kinase activity in glioma cells, and the inhibition was shown not to be d u e to an interaction of the tetraamine with the regulatory subunit of the enzyme (Bachrach et al., 1978; Bachrach, 1980b). Addition of any of the three chief polyamines has been shown to cause a decrease in cAMP concentrations in cultured glioma or neuroblastoma or neuroblastoma x glioma hybrid cells, either unstimulated or stimulated with hormones or drugs (such as norepinephrine, isoproterenol, PGEI, adenosine, IBMX), which are well-known agents for inducing cAMP accumulation inside the cells (C16 e t al., 1979). It is of special interest that a decrease occurred when the exogenous polyamine was added even at low concentrations in the range of those found in physiological fluids (Cl6 e t aZ., 1979). Paradoxically, polyamines at higher concentrations caused a slight increase in the intracellular cAMP levels of the cultured neural neoplastic cell lines (C16 e t al., 1979). Last, a report shows that spermidine and spermine and, to a lesser extent, putrescine are effective inhibitors of the activity of specific cCMP phosphodiesterase obtained from leukemic L1210 cells (Bloch and Cheng, 1979). Friend erythroleukemia cells are a relatively pure population of virus-transformed mouse hematopoietic cells. These cells can be induced by a variety of chemical agents with different biological properties to differentiate to orthochromatic or polychromatic normoblasts and are a suitable experimental system for studying the biochemical events involved in cell differentiation. Interestingly enough, ODC activity can be rapidly induced in this cell line by some inducers of differentiation, such as dimethyl sulfoxide (Tsiftsoglou and Kyriakidis, 1979; Gazitt and Friend, 1980). The ODC induction was observed when the cell differentiation process was blocked or when the inducers were added to cell lysates (Tsiftsoglou and Kyriakidis, 1979). However, the cell differentiation process caused by the inducers in the Friend erythroleukemia cells appears to be not at all mandatory or causal for ODC induction, since some potent inducers, like actinomycin D or aminonucleoside of puromycin, do not stimulate ODC (Gazitt and Friend, 1980). Two final conclusions seem to us to be appropriate. First, the induction of O D C activity appears to be a common and easily observable phenomenon in cultured neoplastic cells, in striking contrast with what has been observed in uivo in neoplastic organs and in organs undergoing chemical carcinogenesis (Scalabrino e t al., 1978). Second, the mediation of CAMP claimed to be a general and possibly obligatory step in ODC induction appears, on the basis of the studies carried out with in uitro systems, to be so in some instances, but to be totally

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GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

extraneous in some others, and this is in quite good agreement with what has emerged from in vivo studies of ODC inducibility.

D. EFFECTSO F

INFECTION WITH

NONONCOGENICVIRUSES

Infection of HeLa cells with vaccinia virus brought about both quantitative and qualitative changes in ODC activity inside the cells, as was demonstrated by Hodgson and Williamson (1975). The ODC was induced and rose quickly following viral infection and, what is even more important, the mean K , value for ODC was significantly lower in infected than in uninfected cells (Hodgson and Williamson, 1975). On the contrary, at later postinfection times the biosynthesis of all the polyamines, including cadaverine, was greatly reduced, but not completely inhibited, in HeLa cells infected with vaccinia virus (Lanzer and Holowczak, 1975). Substantially the same thing was observed in KB cells infected with type 5 adenovirus, at long intervals after infection (Pett and Ginsberg, 1975). This time course for polyamine biosynthesis and concentration in neoplastic cells infected with nononcogenic viruses, with first an increase during early infection and then a decrease at late postinfection times, was also seen in Ehrlich ascites tumor cells infected with mengovirus (Egberts et al., 1977). By way of conclusion, let us tentatively compare the polyamine response of neoplastic cells infected by nononcogenic viruses with the responses observed in normal cells undergoing neoplastic transformation by oncogenic viruses. We can state that (a) there is an increase in cell polyamine biosynthesis immediately after the infection in both types of viral cell infection; (b)there is a clear dichotomy in late phases of postinfection time between the two types of viral cell infection, since the cell polyamine biosynthesis remains at high levels in the neoplastic viral transformation process (see Section IV, Part I, Vol. 35) and decreases progressively after nontransfonning infection of neoplastic cells by nononcogenic viruses.

E. MISCELLANEOUS EFFECTSO F POLYAMINES Most reports on the effects of polyamine addition to cultures of some neoplastic cell lines deal with protein synthesis and cell proliferation. Spermine stimulated poly(UG)-dependent phenylalanine incorporation in a subcellular protein-synthesizing preparation obtained from L1210 mouse ascites leukemic cells, and the stimulation was beyond

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19

that achieved with optimal magnesium concentrations, suggesting that spermine may act as more than merely a substitute for magnesium (Ochoa and Weinstein, 1964). All three chief polyamines (with spermine the most effective) had stirnulatory effects on tRNA methylases in extracts of L1210 cells (Hacker, 1973). Exogeneous spermidine and spermine stimulated the incorporation of orotic acid into RNA and considerably decreased the degradation of the newly synthesized RNA in Ehrlich ascites cells (Raina and Janne, 1968; Khawaja and Raina, 1970). The presence of spermine was essential for the translation in a cell-free system derived from wheat germ of tyrosine aminotransferase mRNA from hepatoma cells (Rether et al., 1978). Again, spermine could partially substitute for soluble factors present in dexamethasoneinduced hepatoma tissue culture that stimulate in a homologous cellfree system the translation of mRNA coding for tyrosine aminotransferase (Beck et al., 1978). In Walker 256 carcinosarcoma cells, putrescine and spermidine preserved the ultrastructural morphology of all nuclear structures, including the nucleolus (Busch et al., 1967). In HeLa cells, polyamines were present in abundant quantities in the chromosome cluster region (Goyns, 1979) and have been shown to stimulate the nuclear synthesis of the histone Hl-poly(ADP-ribose) complex (Byrne et al., 1978).The intercellular adhesiveness of HeLa cells harvested from densityinhibited suspension cultures was markedly enhanced by the addition of putrescine to the medium in which the cells were resuspended (Deman and Bruyneel, 1977). In contrast, the diamine did not modify the mutual adhesiveness of cells harvested from fast-growing cultures (Deman and Bruyneel, 1977). Polyamines have been found also to have some inhibitory effects on cultured neoplastic cells. Spermine depressed protein synthesis in Walker 256 carcinosarcoma cells (Goldstein, 1965). This tetraamine is distinctly cytotoxic for different hepatoma cell lines, and the effect was noticeably enhanced by the presence of fetal calf serum in the growth medium (Katsuta et al., 1975). Among the cytotoxic metabolites released from rat ascites hepatoma cells into culture fluid, some closely resembled spermine in chemical nature (Katsuta et al., 1974). Spermidine, putrescine, and cadaverine all inhibited replicative DNA synthesis in mouse ascites sarcoma cells (Seki et al., 1979).The addition of spermidine or spermine to the medium inhibited the growth of cultured human meningioma cells, whereas putrescine had a slight opposite effect (Duffy et al., 1971). Granulocytic chalone, but not the polyamines, inhibited [3H]TdR uptake in rat chloroleukemia cells in short-term cultures (Foa et al., 1979). This result favors the idea that

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this activity of granulocytic chalone does not depend on its possible polyamine content. The reciprocal connections between synthesis of polyamines and that of 5’-methylthioadenosine (MTA) were evidenced in a human leukemic cell line lacking 5‘-methylthioadenosine phosphorylase (Kamatani and Carson, 1980). The addition of spermine or spermidine markedly depressed the synthesis of MTA, whereas the addition of MTA stimulated putrescine synthesis at low concentration or inhibited it at high concentrations (Kamatani and Carson, 1980). Exogenous MTA also depressed the intracellular levels of spermine in leukemic cells but not, very surprisingly, those of spermidine (Kamatani and Carson, 1980), although MTA is a well-known inhibitor of both spermidine and spermine synthetases (see Section I,D, Part I, Vol. 35). Nevertheless, these relationships between polyamine and MTA metabolisms in neoplastic cells await further experimental elucidation. Finally, spermine precipitated a cell-surface protein, fibronectin, from the culture medium into which it had been secreted by a human rhabdomyosarcoma cell line (Vuento et d.,1980). This observation raises the possibility that polyamines have a role in the deposition of fibronectin in vivo. This is of mounting interest, since many types of malignantly transformed cells, unlike the normal adherent cells, generally deposit small amounts of the surface fibronectin in the pericellular matrix, and this scarcity has been causally correlated with some malignant behavioral properties, which have been overcome by the addition of fibronectin to cultures of tumor cells. Whether elevated polyamine production and secretion (when present) and fibronectin scarcity are interconnected with other factors in determining the well-known poor adhesiveness of neoplastic cells, remains hypothetical, speculative but very attractive. II. Polyamines in Human Oncology

Theoretically, the need to diagnose and subsequently to locate a tumor as early as possible has always been considered to be a key goal for antineoplastic therapy. The expectation that this would be possible was kept alive by new findings, in both experimental and clinical oncology, which showed that certain kinds of neoplasias produce unusual metabolites. Many tumor-cell products, collectively called neoplastic markers,” or “tumor-associated markers” or “oncodevelopmental markers,” have been identified in blood, effusions, urine, and cerebrospinal fluid of tumor-bearing patients and in neoplastic tissue extracts. “

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Measurements of these products have been employed extensively in clinical medicine for both initial diagnosis of neoplasia and monitoring tumor recurrence after different types of therapy. In fact, it was hoped that changes in the amounts of these “markers” in one or more of the different physiological fluids of cancer patients or in the neoplastic tissue could be used to reflect changes in the body’s tumor burden, since these products can be either secreted into the surrounding milieu or kept within the neoplastic cells. The most widely employed “markers” for neoplastic growth and tumor dedifferentiation or differentiation are listed in Table I. As new metheds for the measurement of tumor “markers” were introduced and became ever more sensitive, and clinical studies ever larger, the expectation that these “markers” would be of key importance or, at least, very useful for early diagnosis of neoplasia did not fully materialize. In fact, elevated levels of “ markers” were observed in a variety of nonneoplastic diseases, and, conversely, some neoplasms were not accompanied by any known marker.” Some “markers” have even been found in a percentage of normal healthy adults. In addition, some of these “markers” are also found in the earliest stages of human development, and this association has led to the widely accepted practice of referring to most neoplastic “markers” as oncofetal proteins and antigens (Sell, 1980). The divergence between expectation and practical results was explained after extensive studies of the biochemistry of cancer, which have taught as that tumor cells do not synthesize tumor-specific substances, i.e., substances never found in normal cells at any step in their differentiation (Wolf, 1979a). What is characteristic of tumor cells is that they either express certain normal gene information at the wrong time or in the wrong place or in the wrong amount, or completely fail to express some normal genes (Wolf, 1979a,b). Moreover, during recent decades, it has become ever clearer that neoplasia is not one single type of disease, but a group of very many diseases, each utterly different from the others from the clinical and biochemical points of view, with the only common features that they are lethal to the host and have a cell growth type that is invasive and can never b e stopped definitively. Some frequently found discrepancies between the amount of tumor “marker” present and the degree of growth of the neoplasm must be connected with phenotypic expression of these “markers,” which varies from cell to cell within the neoplasm. In fact, quantitative and qualitative variations in the production of “markers” may occur during the natural course of the malignancy. In other words, during the development of a tumor from preneoplasia to early neoplasia to advanced ‘I

MAIN DIFFERENT BIOCHEMICAL AND

TABLE I “MARKERS”OF NEOPLASTIC GROWTH AND TUMOR DEDIFFERENTIATION DIFFERENTIATION USED I N CLINICAL ONCOLOGY

IMMUNOLOGICAL OR

Products Acute-phase reactant proteins (APRPs): a,-Antitrypsin, a,-antichymotrypsin, ceruloplasmin, C-reactive protein, haptoglobins, fibrinogen Chromosomal abnormalities: Ph’, 13 q-, 14 q+ Cyclic nucleotides: CAMP, cGMP, ratio CAMP: cGMP Enzymes or isozymes: Leucine aminopeptidase, y-glutamyl transferase, copper oxidase, creatine kinase BB, histaminase (DAO), muramidase, galactosyl transferase 11, lysozyme, ribonuclease, arylsulfatase A, reverse transcriptase, terminal deoxynucleotidyl transferase (TdT), superoxide dismutase. Glycolytic isozymes: (a) glucose phosphate isomerase, (b) aldolase (shifting vs A form), (c) LDH (shifting in the isoenzyme pattern from LDH-1 to LDH-5 part of the isoenzyme spectrum). Phosphohydrolases: (a) acid phosphatase, (b) alkaline phosphatase, (c) 5’-nucleotidase Hormones, isohormones, fragments or catabolites of hormones: Ectopic production of hormones (paraneoplastic syndromes): ACTH, gonadotropins, ADH, PTH, ILA, TSH, erythropoietin, MSH, HGH, HPL, HCG, PL, PGA, PGE, CT; catechqlamines, metanephrine, vanilly1 mandelic acid; 5-HIAA, 5-HT, 5-HTP, bradykinin.

References Cooper and Stone (1979) Purtilo et al. (1978) Pardee et al. (1978); Pastan et al. (1975) Bodansky (1975); Bollum (1979); Fishman (1974); Fishman and Singer (1975); Goldberg (1979); Kaplan (1972); Oberley and Buettner (1979); Ruddon (1978); Schapira (1973, 1978); Uriel (1975, 1979); Weber (1977);Wolf (1979b); Yam (1974)

Hall (1974); Ode11 and Wolfsen (1975); Rees and Ratcliffe (1974); Ruddon (1978); Seyberth (1978); Sherwood and Could (1979); Wolf (1979b)

Immunoglobulins: Homogeneous (monoclonal) immunoglobulins (M components); Bence Jones proteins ( K or A light chains); abnormal or incomplete heavy chains: (a) y-chain subclasses (yl, yz. y3, y4) of IgG; (b) a-chain subclasses ( a l , a z )of IgA; (c) p-chains Miscellaneous proteins: Fetal hemoglobin, EDC1, milk casein. Placental and pregnancy proteins: (a) SP, pregnancy-associated a,-glycoprotein (aZPAG); (b) SP, pregnancy-specific &glycoprotein; ( c ) PPTPP8 (ubiquitous tissue) proteins; (d) PPs, placental protein five. Plasminogen activators Oncofetal proteins and antigens: a-FP, CEA, FSA, a2H-ferroprotein; pancreatic oncofetal antigen (POA), p-oncofetal antigen (BOFA), OFA, glial fibrillar acid protein (GFAP)

E3

w

Polyamines and their biosynthetic decarboxylases: Pubescine, spermidine, spermine, ornithine decarboxylase, S-adenosyl-Lmethionine decarboxylase Sterols: Desmosterol (cholesta-5,24-dien-3-P-ol, or 24-dehydrocholesterol)

Bodansky (1975); Solomon (1977); Waldenshom (1976)

Bohn (1980); Ruddon (1978); Rudman et al. (1976, 1977)

Fritsche and Mach (1975); Lehman (1979); Loewenstein and Zamcheck (1977); Martinet al. (1976); Ruoslahti and Seppala (1979); Seidenfeld and Marton (1979b); Sell and Becker (1978); Uriel (1975, 1979); Wikstrand and Bigner (1980) Bachrach (1976a); Janne et al. (1978); Milano et al. (1980); Russell (1977); Russell and Durie (1978); Savory and Shipe (1975); Scalabrino et al. (1980); Seidenfeld and Marton (1978, 1979b) Seidenfeld and Marton (197913);Wikstrand and Bigner (1980)

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GIUSEPPE SCALARRINO AND MARIA E . FERIOLI

metastatic tumor, heterogeneous subvariant cell populations emerge within single clones. Heterogeneity of tumor cell populations may lead not only to the loss of some “marker(s)” but also to the emergence of new ones (Wolfe, 1978; Wolf, 1979a,b). Last, but not least, it is worth mentioning here that some naturally occurring cell labels, such as the glucose-6-phosphate dehydrogenase (G-6-PD) system and the surface-associated immunoglobulins, generally used for identifying different normal cell subpopulations, can also be employed to determine whether a given neoplasm has a single or multiple cell origin, providing an important clue to the initiating event (Fialkow, 1974). The polyamines, although they suffer from the same drawbacks listed above for the other neoplastic “markers,” are widely considered to b e clinically useful “markers” of neoplastic growth and for cancer diagnosis, and particularly for evaluation of the success or failure of an antineoplastic therapy. It is largely accepted to differentiate the different tumor cell “markers” into (a) those produced by dedifferentiation of neoplastic cells (e.g., carcinoembryonic antigen, a-fetoprotein, alkaline phosphatase isozyme) and (b) those produced as a result of overproduction by tumor cells or of the increased tumor cell multiplication (e.g., acid phosphatase, those hormones secreted by specific endocrine-gland neoplasms). Polyamines, for reasons discussed later, have to be included in the second group of neoplastic “markers.” The importance and the clinical significance of polyamines in human oncology have been well reviewed several times by Russell (1973a, 1977), Savory and Shipe (1975), Bachrach (1976b), Cohen (1977), Janne et al. (1978), Russell and Durie (1978), Seidenfeld and Marton (1978, 1979b), Buehler (1980), Durie (1980), and Milano et al. ( 1980). Therefore, we aim here to outline the current “state of the art” about the connections between polyamines and human cancer, together with some recently obtained advances, and to draw particular attention to the use of the levels of activity of the polyamine biosynthetic decarboxylases (PBD) as biochemical indicators of the growth rate, and consequently of the malignancy, of some types of human neoplasias. A. PATTERNS OF POLYAMINES I N HUMANNEOPLASTICTISSUES Hamalainen (1947) made the pioneering observations in this field, systematically screening spermine contents in a great number of organs obtained postmortem from patients who had died of different types of neoplasia. He found an elevated spermine content in the lung

POLYAMINES IN MAMMALIAN TUMORS

25

of a patient who died of lung carcinoma, in the uterus of a patient who died of uterine carcinoma, and in livers, spleens, and bone marrow of two patients who died of leukemia. These observations were subsequently extended to a variety of human malignancies by other authors. Including the more recent reports on the polyamine content of human tumors, it has become ever more evident that there is no general or unique pattern for the polyamine content of human neoplasias. In fact, brain tumor tissues (e.g., neurofibroma, meningioma, glioblastoma, astrocytoma, glioma) have as their particular biochemical feature very high putrescine concentrations in comparison with both gray and white areas of normal human brain (Kremzner, 1970, 1973; Kremzner et al., 1970).On the contrary, the levels of spermidine and spermine in the tumors studied did not greatly differ from levels observed in normal brain, the only exception being high concentrations of spermidine and spermine in astrocytoma and in glioma (Kremzner, 1970, 1973; Kremzner et al., 1970). Additionally, human tumor tissue in vitro and meningioma cells grown in culture actively incorporated [I4C]putrescine into spermidine and spermine, but showed low deaminating activity (Kremzner et al., 1972). The high levels of putrescine in several central nervous system-related tumor tissues have been confirmed (Harik et al., 1978; Harik and Sutton, 1979). Furthermore, it has also been demonstrated that the magnitude of the elevation of putrescine content in the astrocytoma groups is proportional to the degree of malignancy of the tumor as determined by conventional histopathological criteria. A variety of slowly growing and relatively benign intracranial or intraspinal tumors (such as meningioma, cerebellar hemangioblastoma, chordoma, neurofibroma, schwannoma) had low levels of putrescine that in many instances did not exceed the range in samples from normal brain tissue (Harik et al., 1978; Harik and Sutton, 1979). On the other hand, the tissue concentrations of spermidine and spermine varied broadly within normal cerebral cortical samples and within the various tumor types, with no obvious correlation with the degree of malignancy of the tumor (Harik et al., 1978; Harik and Sutton, 1979). Therefore, it can be tentatively concluded that, at least among the astrocytomas, the putrescine level may be a reliable biochemical “marker” not of the tumor per se, but of the degree of malignancy of the tumor. However, amazingly enough, high putrescine levels have been detected in papillary adenocarcinomas of the thyroid, which are the most clinically benign and extremely slow growing of all thyroid malignancies (Matsuzaki et al., 1978). Among the renal cell carcinomas, the concentration of spermidine in

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the poorly differentiated types (grades 3 and 4) was significantly higher than in the well-differentiated types (grades 1 and 2), and in both it was always higher than in normal renal tissue (Matsuda et al., 1978). Thus, in renal carcinomas the concentration of spermidine correlates well with the degree of tumor malignancy ascertained by histopathological known criteria, particularly the nuclear atypia. In the same types of renal tumors, another supposed “marker” for the growth rate of neoplastic cells, i.e., the spermidine : spermine ratio (Russell, 1973b), progressively increases from the normal renal tissue to the poorly differentiated type of renal carcinoma, with the ratio for the well-differentiated type of tumor in the middle (Matsuda et al., 1978). This significant increase in the spermidine : spermine ratio in human renal adenocarcinomas was also confirmed by other authors (Dunzendorfer and Russell, 1978,1979,1980).The concentration of spermidine in neoplastic tissue was significantly higher than in the histologically normal areas of the same kidneys, while the spermine content of the tumor was generally lower than that of normal tissue (Dunzendorfer and Russell, 1978, 1979, 1980). On the contrary, thyroid adenocarcinomas have ratios of spermidine to spermine very close to those found in normal thyroids or those affected by other nonneoplastic diseases (Matsuzaki et al., 1978). The cellular content and secretion of polyamines, in relation to the cell cycle and proliferation kinetics, have been investigated in uitro with cultured cells of Burkitt’s lymphoma (Woo et al., 1979),which is a rapidly growing malignant tumor characterized by a high rate of cell proliferation combined with a high growth fraction. As for the time course of the intracellular contents of polyamines during the growth of Burkitt’s lymphoma cells, large amounts of spermidine and spermine and lower amounts of putrescine were observed during the lag and early exponential growth phases (Woo et al., 1979). This trend was reversed when the cultured cells entered the exponential growth phase and early plateau growth, since spermidine and spermine contents markedly decreased, while the putrescine content tripled (Woo e t al., 1979). The ratio of spermidine to putrescine and that of spermine to putrescine were significantly and positively correlated with both the labeling index and the specific growth rate, whereas there was no significant variation in the spermidine : spermine ratio throughout the growth period (Woo et al., 1979). As for the changes in the cellular polyamine content during the cell cycle, the cell fraction in GI showed a significantly high positive correlation with the intracellular content of putrescine, while negative correlations were calculated for spermidine and spermine (Woo et al., 1979).All these results

POLYAsfINES I N MAMILIALIAN TUMORS

27

seem to suggest that all the chief polyamines actively participate in the process of proliferation of Burkitt’s lymphoma cells. Last, a phospholipid-the so-called malignolipin-containing spermine has been found in some human malignant tumors [e.g., seminoma, gastric cancer, cancer of the colon, uterine cancer, breast cancer (Kosaki et al., 1958)] and in bloods of cancer patients (Kallistratos et al., 1970), but never in normal tissues. This phospholipid contains, in addition to spermine, choline, phosphoric acid, and fatty acids. Although this malignolipin was discovered several years ago, the function and the biological significance of such a compound in tumor development and in tumors of high malignancy remains to be determined and awaits more rigorous demonstration (Bachrach and Ben-Joseph, 1973). B. LEVELSO F THE CHIEF POLYAMINES A N D THEIR CONJUGATED FORMS I N URINES OF NORMAL SUBJECTS A N D OF CANCER PATIENTS Observation of the high polyamine contents in neoplastic tissues at once stimulated looking for increased quantities of these polycationic substances in the extracellular fluids of patients with malignancies. Unlike the neoplastic tissues, the human body fluids generally contain small quantities of polyamines, so that the quantification of these substances in these fluids requires highly sensitive methods. In the last decade noticeable improvements in the assay methods for polyamines have been achieved (Seiler, 1977, 1980). The assay methods most widely used at present for quantitative determinations of polyamines and their derivatives are thin-layer chromatography of dansylated polyamines, automated ion-exchange chromatography, high-pressure liquid chromatography, gas chromatography, and radioimmunological assay. All these methods are sensitive and accurate enough to detect very small amounts of polyamines in both physiological fluids and in biopsy material. Therefore, routine screening for polyamine levels in fluids of human beings with different types of pathologies, whether or not characterized by uncontrolled cell proliferation, is now possible. The data available in the literature on the daily urinary excretion of polyamines and of their conjugated forms by normal subjects are reported in Tables I1 and 111, where the values have been divided into groups according to the units of measurement used by the various authors to express their results. Those data reported by some authors as control levels, but taken from hospitalized patients with nonneoplastic diseases, have deliberately not been reported in Tables I1 and 111,

NORMAL DAILYCONTENTS Unit mgl24-hr U

rmoll24-hr U

pmollkgl24-hr U

POLYAMINES IN HUMANURINE"

N

Putrescine

Spermidine

Spermine

2 50 50 10 5 12 8 6 42 NR 42 50 56 21 8 20 9

2.5 2.7 2 0.53 2.7 2 0.5 2.5 f 0.6 2.0 1.4 0.94 2.2 3.52 0.2-2.84 0.8-6.2 3.52 4.21 f 0.41 0.89 1.52 0.98 0.49 1.6 2 0.4 1.6 (9) 1.57 2 1.05 21.9 2 7.6 9.8 2.0 0.5 0.38 2 0.017 0.2 0.4 0.38 2 0.17 0.7

2.7 3.1 0.56 3.1 2 0.6 2.4 f 0.4 1.5 1.3 0.86 1.6 2.44 0.36-2.1 0.9-3.9 2.44 1.12 0.11 0.53 0.83 4.57 2 1.02 0.2 2 0.04 0.3 (7) 0.51 0.16 8.4 f 2.1 7.6 2 2.5 0.2 0.1 f 0.003 0.12 0.11 0.11 f 0.04 1.5

2.5 3.4 2 0.67 3.4 2 0.7 0.4 2 0.2 <0.4 0.4

12 12 5 13 28 35

44 pg/ml U

TABLE I1 OF THE CHIEF

50 8

*

*

*

*

*

-

0.4 2.59 0.0-0.87 1.0-4.2 2.59 3.4 2 0.67 0.14 0.20 0.31 f 0.09 2.1 2 1.0 2.0 (7) 0.71 f 0.57 2.5 f 1.2 2.5 2 0.8 0.07 0.01 f 0.002 0.02 0.01 0.01 f 0.006 -

Remarks

Reference Russell (1971) Russell et d. (1971a) Schimpff et QZ. (1973) Marton et QZ. (1973a) Marton et ~ l (1973b) . Gehrke et QZ. (1973) Gehrke et ~ l (1973) . Gehrke et QZ. (1974) Kessler et aZ. (1974) Tormey et QZ. (1975) Sanford et QZ. (1975) Lipton et 01. (1975, 1976) Fujita et QZ. (1976) Adler et QZ. (1977) Makita et QL. (1978) Brown et QL. (1979) Abdel-Monem and Ohno (1978) Abdel-Monem et QZ. (1978) Rattenbury et QZ. (1979) Rattenbury et ~ l (1979) . Swendseid et QZ. (1980) Waalkes et QZ. (1975a) Waalkes et QZ. (1975b) Waalkes et QZ. (1975b) Waalkes et aZ. (1975b) Woo et QZ. (1978) Veening et QZ. (1974)

10 12 15 16 NR NR 12 13 10 6 5 10 11 NR 7 61 56 9 10 NR

pmol/mg CR mglg CR nmol/mgCR pmol/g CR pmol/kg/24-hr U mg/24-hr U pmollg CR mglg CR

28 21 10 61

3.58 f 0.99 1.8 2.5 2 0.13 2.1 f 0.62 1.31 1.79 3.0 2 0.84 3.5 2.5 f 0.8 2.73 2 0.59 2.09 2 0.57 2.9 3.4 1.4 1.44 1.68 2 0.62 125.3 f 17.7 13.5f 2.16 0.89 f 0.76 0.45 0.02 f 0.003 0.55 0.93 f 0.50 0.86 f 0.60

1.3 1.7 2 0.10 1.2 f 0.18 0.92 1.02 2.2 f 0.44 2.6 1.1 f 0.5 1.56 2 0.42 1.79 2 0.49 1.4 2.1 0.5 0.81 1.32 f 0.41 20.3 f 2.4 6.3 f 0.35 0.67 f 0.57 0.28

0.5 f 0.24 0.5 f 0.06 0.04 2 0.007 0.27 0.19 0.51 0.0-5.5 0.05 f 0.007 0.06 f 0.01 0.14 f 0.08 8.4 4.5 0.18 0.18 f 0.29 46.4 f 12.5 0.9 f 0.15 -

Cadaverine Cadaverine Cadaverine Cadaverine

-

-

-

In(PUT U/g CR) = 0.013(age + 3/4) - 0.031 ln(age + 3/4) ln(SPD U/g CR) = - 1.454 - O.O12(age + 3/4) - 0.071 In(age + 3/4) ln(SP U/g CR) = 4.787 - 0.027(age + 3/4) - 0.013 ln(age + 3/4) ~~

~

Rennert et al. (1976a) Russell et al. (1975) Townsend et al. (1976) Russell (1977); Durie et nl. (1977a) Nishioka et al. (197813) Nishioka et al. (1978b) Heby and Andersson (1978a) Osterberg et al. (1978) Russell et al. (1978) Russell et al. (1979) Russell et al. (1979) Tsuji et al. (1975) Tsuji et al. (1975) Slanina et al. (1979) Makita et al. (1978) Fujita et al. (1980) Fujita et al. (1976) Proctor et al. (1979) Berry et al. (1978b) Berry et al. (197813) Waalkes et al. (197%) Adler et al. (1977) Berry et al. (1978b) Fujita et al. (1980) Rudman et al. (1979) Rudman et al. (1979) Rudman et al. (1979) ~~

"The results reported are expressed as means alone, or means 2 SE, or means 2 SD (when specified). N, number of subjects; NR, not reported; FP, free polyamine; TP, total polyamine; M, men; W, women; C, children; U, urine; CR, creatinine; kg, kilogram of body :veight; R, range; PUT, putrescine; SPD, spermidine; SP, spermine.

TABLE 111 NORMALDAILYCONTENTS OF THE CONJUGATED FORMS OF POLYAMINES IN HUMANURINE" Unit

N

Ac-PUT

NI-Ac-SPD

NMcSPD

pmo1/24-hr U

3 3 9 12 5 5 10-11 10 9

2.10 11.7 ? 1.5 14.2 22.0 t 3.9 21.0 t 5.6 N-Ac-S PD N-Ac-SPD Ac-cadaverine

0.382 0.382 2.9 t 0.6 2.9 6.6 ? 2.4 4.7 ? 1.4 5.4 M; 7.1 W 3.48 t 2.62 C; 0.62 1.9 .9

0.306 0.306 2.84 f 0.5 2.8 5.3 ? 1.2 4.1 t 1.3

pmol/mg CR pmollg CR pmo1/24-hr U

*

Remarks

-

SD, M SD, W

Reference Abdel-Monem et al. (1975b) Abdel-Monem and Ohno (1977b) Abdel-Monem and Ohno (1978) Abdel-Monen et al. (1978) Seiler and Knodgen (1979b) Seiler and Knodgen (197913) Tsuji et al. (1975) Berry et al. (197813) Abdel-Monem and Ohno (1978)

"The results reported are expressed as means alone, or means ? SE, or means t SD (when specified). N, number of subjects; M, men; W, women; C , children; U, urine; C R , creatinine; Ac-PUT, acetylputrescine; N1-Ac-SPD, N*-Ac-SPD, N'- and N 8 acetylspermidine; N-Ac-SPD, N-acetylspermidine ; Ac-cadaverine, acetylcadaverine.

POLYAMINES IN MAMMALIAN TUMORS

31

since it has been well demonstrated that urinary levels of one or more polyamines can be abnormal in many nonneoplastic diseases (e.g., cystic fibrosis, infectious diseases, psoriasis, anemias of different etiology, rheumatoid arthritis, systemic lupus erythematosus, polymyositis, cardiovascular diseases, pulmonary tuberculosis, hepatitis, hereditary muscular dystrophies, cystinuria, some inborn errors of metabolism, some inborn defects of renal transport of amino acids) (Kessler et al., 1974; Dreyfuss et al., 1975; Waalkes et al., 1975b; Durie et al., 1977a; Berry et al., 1978b; Janne et al., 1978; Russell and Durie, 1978; Rudman et al., 1980). Even in physiological states, such as normal pregnancy (Russell et al., 1978), or during therapeutic treatments, such as in growth hormone (GH)-deficient children after GH treatment (Rudman et al., 1979), elevations of total polyamines or of at least one or two polyamines have been observed. It appears from Tables I1 and 111 that the values for daily urinary polyamine excretion by healthy humans are quite erratic and sometimes conflicting. It is difficult to compare the normal values reported by the different authors, even within each group of values, for several reasons. First of all, the authors used different units of measurement to calculate and express the daily amounts of urinary excretion of polyamines and of their conjugated forms. Second, another fundamental source of the great variability of the values in Tables I1 and 111, is the large number of different assay methods employed, beginning with the not entirely specific methods of the earliest reports to the most sophisticated and most reliable of the most recent reports. Third, there are also some physiological sources of the variability in the normal data published so far. One of these is the age range of healthy volunteers, since this range is quite wide in most of the reports. However, an influence of age on the amounts of the urinary polyamines excreted per day has been demonstrated and carefully investigated in humans, from 0 to 70 years of age (Rudman et al., 1979).The fact that the determinations of polyamines were usually carried out on the urine of only one day seems to be of minor importance, since normally the excretion of polyamines is quantitatively quite constant, with only small variations from one day to another (Waalkes et al., 197513).Another source of variability is the sex of the control subjects. The majority of the mean values published by the authors were obtained by averaging the levels of polyamines for men and women. However, women have been demonstrated to excrete more putrescine than men, and men have urinary spermine contents greater than those of women (Tsuji et al., 1975; Waalkes et al., 1975b; Nishioka et al., 1978b). However, a report has confirmed this influence of the sex on the daily

32

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

urinary excretion of putrescine, whereas higher values of excretion for spermine were found in woman than in man (Beninati et ul., 1980).No difference between the sexes was found in urinary elimination of spermidine and cadaverine (Tsuji et al., 1975; Waalkes et al., 1975b; Nishioka et al., 1978b; Beninati et al., 1980). As for the conjugated forms of polyamines, it has been shown that men excrete N ' acetylspermidine and N8-acetylspermidine in higher quantities than women (Seiler and Knodgen, 1979), but there is no significant variation between the sexes in daily excretion of acetylputrescine (Seiler and Knodgen, 1979). Interestingly, the urinary excretion of all three chief polyamines has been demonstrated to be enhanced during menstruation, and sometimes it remained increased during the early follicular phase (Osterberg et al., 1978). This increase usually has been connected with endometrial necrosis (Osterberg et d.,1978),since several reports have shown that the extracellular polyamine contents are very often augmented as a consequence of cell death (Heby and Anderson, 1978b; Woo et al., 1979), though there may also be some effects of the concomitant changes in hormonal levels. In 1971, simultaneous studies of Russell (1971), Russell et al. (1971a,b), and Bremer et al. (1971)demonstrated elevated daily excretion of some polyamines by cancer patients or by patients with cystinuria. This immediately emphasized that it is not possible to consider an increase in urinary polyamine excretion to be a biochemical feature characteristic of the neoplastic state. Those reports by Russell and her co-workers, mainly concerning hematologic tumors and some solid tumors, raised the question of whether analysis of polyamines in urines of cancer patients would be useful diagnostic tools for cancer detection and aroused much interest in this field. Since that time, many other authors have carried out urinary polyamine analysis in cancerous subjects and have confirmed that the polyamine content is frequently high. Nevertheless, the hopes stirred up by the initial findings have been partially disappointed, because later findings, particularly in more recent years, have clearly shown that the magnitude of the increases in urinary polyamine levels in cancer patients differ not only in relation to the type of neoplasia but also within the same type of neoplasia. Moreover, the extent of the increases are sometimes no larger than those observed in other nonneoplastic ill subjects, and, what is more, there are even some reports in which no differences between the daily amounts of excreted polyamines in patients with cancer and normal persons were observed (Gehrke et al., 1973, 1974). Last, but not least, it has also been found that the percentage of pa-

POLYAMINES IN MAMMALIAN TUMORS

33

tients with localized malignant tumors who show an elevation of urinary polyamines was not much different from that of patients with benign tumors (Lipton et al., 1976). In the following listing, the different types of human neoplasms are grouped on the basis of the location of the primary tumor. Then, despite the aforementioned drawbacks, for each group of tumors we will cite the papers published so far, showing enhancements of daily urinary excretion of one or more polyamines in some patients with the various tumors. Those papers in which the types of tumors were not clearly specified, have been deliberately omitted from the following list. 1. Neoplasias of the digestive system and associated glands. Tumors of the esophagus (Lipton et al., 1975, 1976; Waalkes et al., 1975b; Fujita et al., 1976), the stomach (Dreyfuss et al., 1975; Tsuji et al., 1975; Waalkes et al., 1975b; Fujita et al., 1976) or the small bowel (Waalkes et al., 1975b), the colon (Dreyfuss et al., 1975; Waalkes et al., 1975b; Fujita et al., 1976; Durie et al., 1977a), the rectum (Russell, 1971; Russell et al., 1971a; Kessler et al., 1974; Dreyfuss et al., 1975; Fujita et al., 1976; Lipton et al., 1976; Nishioka et al., 1978b), the liver (Abdel-Monem et al., 1975b; Waalkes et al., 1975b; Fujita et al., 1976; Abdel-Monem and Ohno, 1977a,b, 1978), the gallbladder (Fujita et al., 1976), the bile duct (Fujita et al., 1976), and the pancreas (Dreyfuss et al., 1975; Waalkes et al., 1975b; Fujitaet al., 1976; Lipton et al., 1976). 2. Neoplasias of the respiratory system. Malignant lung tumors (Marton et al., 1973a; Kessler et al., 1974; Dreyfuss et al., 1975; Lipton et al., 1975, 1976; Waalkes et al., 1975b; Fujita et al., 1976; Heby and Anderson, 1978a; Woo et al., 1980). 3. Neoplasias of the female reproductive system. Cancer of the uterus (Russell et al., 1971a; Fujitaet al., 1976; Lipton et al., 1976), the ovaries (Russell, 1971; Russell et al., 1971a,b; Schimpff et al., 1973), and the vagina (Lipton et al., 1976). 4. Neoplasias of the male reproductive system. Malignant tumors of the prostate (Dreyfuss et al., 1975; Fair et al., 1975; Sanford et al., 1975; Waalkes et al., 1975b; Durie et al., 1977a) and the testicles (Russell et al., 1971a; Marton et al., 1973a,b; Schimpff et al., 1973; Russell and Russell, 1975; Sanford et al., 1975; Durie et al., 1977a). 5. Neoplasias of the urinary system. Renal tumors, such as hypernephroma or carcinoma (Dreyfuss et al., 1975; Sanford et al., 1975; Waalkes et al., 197513; Lipton et al., 1976), and bladder carcinomas (Dreyfuss et al., 1975; Sanford et al., 1975; Lipton et al., 1976; Durie et al., 1977a; Heby and Anderson, 1978a).

34

GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

6. Neoplasias of the hematopoietic system. Generally speaking, the greatest elevation in urinary polyamine levels has been found in hematologic malignancies: leukemias (Russell, 1971; Russell et al., 1971a,b, 1975; Gehrke et al., 1973; Schimpff et al., 1973; Tsuji et al., 1975; Fujita et al., 1976; Heby and Andersson, 1978a), lymphosarcoma (Russell, 1971; Russellet al., 1971a,b; Gehrke et al., 1973; Dreyfuss et al., 1975; Heby and Andersson, 1978a), Hodgkin’s disease (Russell, 1971; Russell et al., 1971a; Denton et al., 1973a; Gehrke et al., 1973; Marton et al., 1973a,b; Heby and Anderson, 1978a), reticulum cell sarcoma (Russell et al., 1971a,b; Gehrke et al., 1973; Tsuji et al., 1975; Fujita et al., 1976; Heby and Anderson, 1978a), multiple myeloma (Gehrke et al., 1973; Dreyfuss et al., 1975; Fleisher and Russell, 1975; Russell and Russell, 1975; Russell et al., 1975; Tsuji et al., 1975; Durie et al., 1977a; Heby and Andersson, 1978a), various forms of nonHodgkin’s lymphomas (including Burkitt’s lymphoma) (Gehrke et al., 1973; Schimpff et al., 1973; Russell et al., 1975; Waalkes et al., 1975a,b). 7. Neoplasias of the integumentary system. Cutaneous malignancies: melanoma (Russell et al., 1971a; Kessler et al., 1974; AbdelMonem et al., 1975b; Fleisher and Russell, 1975; Lipton et al., 1975; Rodermund and Moersler, 1975; Townsend et al., 1976; Abdel-Monem and Ohno, 1977b; Durie et al., 1977a; Gittins and Cooke, 1978; Heby and Andersson, 1978a), basal cell epithelioma (Rodermund and Moersler, 1975), mycosis fungoides (Rodermund and Moersler, 1975), and Sezary syndrome (Rodermund and Moersler, 1975). 8. Neoplasias of the mammary gland. Urinary polyamines are high in only a small percentage of patients with breast cancer (Russell et al., 1971a; Kessler et al., 1974; Fleisher and Russell, 1975; Lipton et al., 1975, 1976; Tormeyet al., 1975,1980; Tsujiet al., 1975; Waalkes et al., 1975b; Durie et al., 1977a; Gehrke et al., 1977; Heby and Anderson, 1978a; Nishiokaet al., 1978b; Wooet al., 1978). This is in keeping with the biological features of these types of neoplasia, which usually have small growth fractions and slow growth rates. 9. Neoplasias of the central nervous system (CNS). There are very few reports on patients with CNS tumors: neuroblastoma (Wall, 1971), glioblastoma (Dreyfuss et al., 1975), and astrocytoma (Waalkes et al., 1975b). 10. Neoplasias of the endocrine system. Thyroid carcinoma, the only one studied (Abdel-Monem et al., 1975b; Abdel-Monem and Ohno, 197713). 11. Neoplasias of bone. Osteogenic sarcomas (Russell, 1971; Russell et al., 1971a; Tsuji et al., 1975; Waalkes et al., 1975b; Heby and Andersson, 1978a).

POLTAMINES I N M A M M A I J A N TUMORS

35

From the survey of the vast literature on this topic, four main conclusions can be drawn.

1. The quantities of polyamines excreted by cancerous patients do not always correlate directly with the growth rate of the tumor. Very frequently, patients with Burkitt's lymphoma (a rapidly proliferating neoplasia with large growth fraction) or with certain other hematologic malignancies excrete very large amounts of polyamines, but a high percentage of patients with carcinoma of the colon (a tumor with a small growth fraction and a slow growth rate) also have increased polyamine excretion. 2. The remarkably high incidence of false-negative values (i.e., the number of patients with advanced malignant disease who have normal levels of urinary polyamines) and the remarkably high incidence of false-positive patients (i.e., the number of patients with diseases other than cancer who have elevated urinary polyamine contents) suggest that the determination of urinary polyamine levels only in cancer patients is of little, or perhaps even no, importance for cancer diagnosis, particularly for early cancer diagnosis. 3. Nevertheless, the satisfactorily high percentage of cancerous patients (with a rather wide variety of types of advanced neoplasias with both scarce and widespread metastases), who have elevated urinary levels of one or two of the chief polyamines causes us to feel that when polyamines are elevated, this can be viewed simply as a general epiphenomenon of neoplastic growth. 4. It is well known that in normal human urines, unlike in the contents of the mammalian cell, free polyamines are present in lesser amounts than conjugated polyamines, which are mostly acetylated derivatives. In turn, among the acetylated forms of polyamine, the acetylspermidines and acetylputrescine are quantitatively predominant over the acetylated forms of spermine and cadaverine. These products have been found in cancer patients too (Denton et al., 1973b; Walle, 1973; Abdel-Monem et al., 1975b, 1978; Tsuji et al., 1975; Abdel-Monem and Ohno, 1977a,b, 1978; Rosenblum, 1980). Furthermore, an interesting series of papers by Abdel-Monem and his co-workers dealing with the acetylated forms of spermidine excreted by normal and cancer patients have demonstrated that the great majority of cancer patients have a higher urinary N'-acetylspermidine : N8-acetylspermidine ratio than normals, and the elevation of the ratio is essentially due to an increase in the amount of N'-acetylspermidine excreted (Abdel-Monem et al., 1975b, 1978; Abdel-Monem and Ohno, 1977b, 1978). Whether the increase of this molecule in urines of cancerous subjects is due to a higher rate of formation or to lesser

36

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

degradation, relative to NB-acetylspermidine, remains to be established. Moreover, the specificity of the increase in that ratio for cancer patients must be confirmed, since there are no analogous clinical studies in patients with diseases other than cancer. The urinary levels of acetylputrescine and acetylcadaverine have also been found to be higher in cancer subjects than in normal ones (Abdel-Monem et al., 1978), although this finding also awaits further confirmation in noncancer patients before it can be claimed as a characteristic of the neoplastic state. All together, these results indicate that the specific abnormality, if any, of the urinary profiles of all the polyamines [including the acetyl derivatives and lY3-diaminopropane (Walle, 1973; Heby and Andersson, 1978a; Slanina et al., 1979)] in cancer patients might consist in a shifting of the quantitative ratios between the various molecules rather than in mere quantitative elevation. Other fundamental problems in clinical oncology are to determine as exactly as possible the stage of the tumor, the prognosis, the response to therapy, and the remission-relapse status. On present evidence, the use of polyamine profiles in urines of cancer patients has given the most promising results in this particular field, especially in short-term evaluation of the efficacy of therapy and in the assessment of the activity status of the neoplasm. In fact, several reports have shown that a successful chemotherapy or immunotherapy or the surgical removal of a tumor very frequently brings about a marked decrease in the urinary polyamine levels of cancer patients in the days and weeks after treatment, whereas the polyamine levels were changed much less or not at all in patients unresponsive to the different types of chemotherapy when the neoplasm recurred or became terminal, or one or more or all of the polyamines rose once again (Russell, 1971; Russell et al., 1971a,b; Denton et al., 1973c; Schimpff et al., 1973; Fleisher et al., 1974; Dreyfuss et al., 1975; Lipton et al., 1975; Sanford et al., 1975; Takeda et al., 1975; Waalkes et al., 1975a,b; Fujita et al., 1976; Townsend et al., 1976; Slanina e t al., 1979; Nissen et al., 1980; Tormey et ul., 1980). However, more detailed and careful studies carried out during or immediately after the beginning of treatment have demonstrated that acute elevations in urinary polyamine levels occur in response to effective chemotherapy, before those levels return to near normality during the remission phase (Denton et al., 1973a,b, 1974; Russell and Russell, 1975; Russell et al., 1975; Waalkes et al., 1975a; Durie et al., 1977a; Russell, 1977; Russell and Durie, 1978).Accordingly, a specific time course for the changes in urinary polyamine values in cancer

POLYAMINES IN MAMMALIAN TUMORS

37

patients responding to effective therapy has been tentatively established (Russell and Russell, 1975; Russell et al., 1975; Durie et al., 1977a; Russell, 1977; Russell and Durie, 1978; Woo et al., 1980).This time course consists, during the first days after the initiation of successful chemotherapy, of a rise of the spermidine levels that is thought to reflect the killing of tumor cells. More in detail, the free spermidine level changed only slightly, while the level of conjugated spermidine was markedly augmented (Rosenblum, 1980). This is generally followed by a return to near normal spermidine values or at least to those existing before therapy. Usually, this decrease in urinary spermidine levels is accompanied by remission of the neoplastic disease. Furthermore, enhancement of urinary putrescine levels in cancer patients after the end of the chemotherapy reflects recruitment of tumor cells into the proliferative compartment, and therefore reflects the tumor load and a recurrence of the neoplastic disease. As for spermine, it was suggested that an abnormal elevation of this tetraamine could reflect cellular aging leading to spontaneous cell loss, i.e., not cell destruction by chemotherapy (Tormey et al., 1980). Thus, in long-term surveillance of cancerous patients, changes in urinary polyamine levels seem very often to correlate with the clinical status of the patient, and monitoring could predict the clinical decline of the patients. However, we still do not know the extent of the contribution of the polyamines produced from tissues, other than the neoplasias that are also affected by cytotoxic agents. Once the urinary spermidine level was held to be a reliable marker of the response to the effective chemotherapy, another clinically useful indicator was introduced for differentiating the patients into nonresponders, partial responders, and complete responders to the different types of chemotherapy. This indicator is the posttreatment :pretreatment spermidine ratio (Durie et al., 1977a; Woo et al., 1978). In nonresponding patients with hematologic or solid malignancies, the mean value of the ratio was found to be around 1.2 and never to exceed 2, whereas the mean values of the ratio for partially or completely responsive patients are between 3 and 4. Nevertheless, in evaluating the real clinical importance of all these urinary parameters connected with polyamines and their conjugated forms in the assessment of the disease status in cancer patients, it must always be borne in mind (a) that marked changes in polyamine excretion may be caused by factors other than anticancer chemotherapy (Denton et al., 1973a; Nishioka et al., 1980);(b)that the clinical malignancy of a tumor is widely thought to be dependent on and connected with not only some biochemical parameters, even though theoretically

38

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

appropriate and suitably evaluating biological aspects of the malignancy of the tumor, but also with other important factors, such as the localization of the tumor, its ability to invade vessels and consequently metastasize, and so forth. Finally, since the urinary polyamine levels, considered alone, are not at all satisfactory markers for neoplastic disease and inadequate for the needs, combinations of urinary polyamine levels plus other relatively tumor-specific molecules present in blood and/or urines (e.g., urinary nucleosides, CEA) might considerably improve diagnostic specificity and the evaluation of tumor changes during treatment (Tormey et al., 1975; Woo et al., 1978; Nishioka et al., 1978a). Combinations of neoplastic “markers” may have remarkable advantages over single “markers,” because different neoplastic “markers” often seem to reflect different biological aspects of the neoplasia.

c. LEVELSOF T H E C H I E F POLYAMINES AND THEIRCONJUGATED

FORMSIN BLOOD, PLASMA, SERUM, FORMED BLOODELEMENTS, BONE MARROWOF NORMALSUBJECTS AND OF CANCER PATIENTS

AND

1. Polyamines in Whole Blood and Its Liquid Parts Determinations of polyamine contents in whole blood, in plasma, and in serum from normal and oncopathic subjects have accumulated since early reports of Tokuoka (1950, 1956), who proposed the cupric carbonate test no longer used (Bachrach and Robinson, 1965) for the diagnosis of malignancy, based on the reaction of this substance with the spermine present in the sera of cancerous patients. The data available in the literature for the levels of putrescine, spermidine, spermine, and cadaverine in whole blood, plasma, and sera of normal men and women are reported in Table IV. In this table, we see once again that the values are quite erratic and sometimes even conflicting. Some of the reasons listed earlier to explain in part the great variability of the polyamine levels in normal urines, e.g., the different units of measure used, the different assay methods employed, the physiological source of variability aforementioned, are valid also to account, at least in part, for the great variability in normal polyamine levels in whole blood, plasma, and serum. Therefore, none of these will b e discussed further here. Some general but quite certain conclusions can be drawn about the levels of polyamines in normal human blood and in its liquid parts.

1. Whole blood has a higher polyamine content than plasma or serum because nearly 95% of the polyamines in whole blood are in the

POLYAMINES I N MAMMALIAN TUMORS

39

blood cells (Cohen et al., 1975, 1976; Lundgren e t al., 1975; Cooper et al., 1976, 1978; Rennert and Shukla, 1978; Saeki et al., 1978). 2 . No remarkable differences have been found between polyamine levels in plasma and serum. This means that the clotting process does not subtract polyamines from plasma. 3. Polyamines in normal sera are present in the unconjugated forms, with a minor part conjugated to a peptide carrier (Seale et al., 1979) or to fibronectin (Roch et al., 1980). As for the acetyl-derivative forms of polyamines present in normal sera, N '-acetylspermidine has been identified, but not NX-acetylspermidine (Smith et al., 1978), unlike in urines. Monoacetylcadaverine and monopropionylcadaverine have also been identified in normal blood (Dolezalova et al., 1978). 4. In normal plasma and serum, there is less spermine than putrescine and spermidine, the only exception being reported by Chaisiri et al. (1979). 5. Like urinary polyamine levels, spermidine and spermine concentrations in whole blood fluctuate in women during the menstrual cycle (Lundgren et al., 1976; Rennert et ul., 1976a; Campbell et al., 1977), but not in men observed over the same length oftime (Lundgren et al., 1976), suggesting the existence of a sex-related hormonal influence on blood spermidine and spermine levels. 6. Among the possible different analytical assay methods for polyamines in the blood, the choice and the reliability of a method depends on what blood compartment is to be assayed. For instance, for serum, there are three independent methods, i.e., high pressure cation-exchange chromatography, radioimmunoassay, and gas chromatography-mass spectrometry, that are highly sensitive and allow polyamine analysis of very small amounts of serum (D. Bartos et al., 1975; F. Bartos et al., 1977; Bonnefoy-Roch and Quash, 1978). Despite the initial promise, the same positive and negative comments listed above apropos the real clinical significance and usefulness of urinary polyamine determinations in human oncology have to be repeated and kept in mind when one is evaluating the real importance of polyamine determinations in whole blood, plasma, and serum of cancer patients in relation to cancer diagnosis and treatment. 1. The frequently observed increases in polyamine levels in blood, plasma, or serum from cancerous subjects are not at all specific for this kind of disease, since other analogous elevations have been recorded in some nonneoplastic illnesses, such as cystic fibrosis (Lundgren et al., 1975; Arvanitakis et al., 1976; Rennert et al., 1976a; Berry et al., 1978a), systemic lupus erytheinatosus (Puri et al., 1978), schizophrenia (Pfeiffer et al., 1970; Dolezalova et al., 1978), sickle cell anemia

TABLE IV NORMALLEVELSOF THE CHIEF POLYAMINES AND THEIRCONJUGATED FORMS IN HUMANBLOOD,PLASMA, AND SERUM' Specimen and unit Whole blood pglml pg/ml d m l Lrdml Wml Pdml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmollml nmollml nmol/ml nmollml Plasma nmol/ml nmol/ml nmol/ml d m g CR nmollml n m o1/ m 1 nmol/ml pm ol/l iter nmollliter nmollliter

N

Putrescine

Spermidine

Spermine

Remarks

13 17

-

0.95 2 0.05 0.97 2 0.04 1.26 0.99 2 0.18 1.0 0.19 1.17 0.57 10.3 2 1.01 8.47 0.56 4.87 -t 0.37 3.87 2 1.29 2.54 5 1.51 3.3 f 0.29 7.07 9.69 6.56 f 0.39

1.4 f 0.12 1.25 2 0.06 0.26 1.27 2 0.48 1.48 0.34 1.10 f 0.27 9.97 f 0.87 7.25 0.82 4.28 2 1.4 4.01 f 1.37 2.60 2 1.25 1.93 2 0.33 4.68 7.08 3.92 f 0.36

W M

50.12 0.07 0.01 0.08 f 0.02 0.29 2 0.1 0.13 f 0.04

S O . 16 0.08 0.02 0.03 0.01 0.01 0.04 0.015 0.20 2 0.10 0.19 2 0.09 0.06 -

3 10 19 NR 14 13 NR 4 30 11 2 22 17 10 20 17 10 37 66 96 3 61 98

-

1.1 f 0.14 -

-

0.21 2 0.02 0.9 0.35 0.9 0.35 0.08 2 0.01 0.23 2 0.1 0.1 2 0.03 -

*

-

0.13 -

* * *

*

-

0.22 200 f 137.7 201 2 116.8

*

* *

-

W M SD M W SD -

-

-

-

SD M, SD W, SD

-

M , SD W, SD

Reference Raina (1962) Raina (1962) Shimizu et al. (1965) Iliev et al. (1968) Iliev et al. (1968) McEvoy and Hartley (1975) Lundgren et al. (1975) Lundgren et al. (1975) Arvanitakis et al. (1976) Chun et al. (1976) Rennert et al. (1976a) Rennert and Shukla (1978) Saeki et al. (1978) Berry et al. (1978a) Cooper et al. (1978) Cooper et al. (1976) Rennert and Shukla (1978) Cooper et al. (1978) Russell et al. (1978) Takami et al. (1979) Chaisiri et al. (1979) Chaisiri et al. (1979) Shipe et al. (1979) Chaisiri et al. (1980) Chaisiri et al. (1980)

*

nmollml nmol/ml Serum nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml nmol/ml

NR 11

0.11 0.03 0.11 2 0.03

0.12 0.03 0.13 2 0.04

NR NR 10 7 7 6 23

0.23 0.09 0.31 0.17

0.32 0.07 0.25 0.33 2 0.09 0.33 0.10 0.39 5 0.06 0.23 t 0.05

pmollml pmollml nmol/ml nmollml nmollml pmol/ml

10 10 2 17 16 23

130 14.7 270 2 23.9

*

-

0.17 t 0.08

*

-

0.51 2 0.06 0.15 0.04 98.9 t 45.2

*

*

*

70 18.4 240 2 30.6 0.78 0.63 2 0.07 0.14 2 0.03 88.5 -+ 47.4

0.03 0.04

* 0.01

" 0.02 -

0.04 0.02 -

*

-

0.12 2 0.008 0.03 t 0.02 20 50

* 9.3

0.33 2 0.08 0 29.1 -+ 26.0

Miscellaneous reportsb Cadaverine In blood 10.9 (24) (pmol/g wet wt.) In serum 0.14 (7) TP; 0.03 (7) F P (nmol/ml) 10 (10) FP; 60 17.33 (10) T P (pmol/ml) N'-Ac-SPD in serum 0.008-0.05 (nmol/ml) FPs in serum 28 2 4.7 (8) (ng/ml) 43.4 2 14.8 (40) (ng/ml) C, SD = 0.206 - 0.008(age + 3/4) - 0.320 ln(age + 3/4) ln(PUT serum) = 0.473 - 0.004(age + 3/4) - 0.142 In(age + 3/4) In(SPD serum) = -1.532 - O.O16(age + 3/4) - 0.296 ln(age + 3/4) ln(SP serum)

*

14.5

SD SD

Takami e t QZ. (1980a) Takami and Nishioka (1980)

SD TP FP SD SD

Marton et d. (1973a) Marton e t ~ l (1973b) . Nishioka and Romsdahl (1974) Saniejima et d. (1976) Samejima et d. (1976) F. Bartos et QZ. (1977) Nishioka et al. (1977, 1978a); Nishioka and Romsdahl (1978) Kai e t d. (1979) Kai et QZ. (1979) Hospattankar et aZ. (1980) Baylin e t d. (1980b) Baylin et al. (1980b) Desser et al. (1980)

FP TP C SD

Dolezalova et ~ l (1978) . Samejima et d. (1976) Kai et d. (1979) Smith et d. (1978) Bartos e t al. (1975) Puri et d. (1978) Rudman et aZ. (1979) Rudman et al. (1979) Rudman et aZ. (1979)

"The results reported are expressed as means alone, or means 2 SE, or means 2 S D (when specified). N, number of subjects; NR, not reported; FP, free polyamine; TP, total polyamine; M, men; W, women; C, children. *N'-Ac-SPD, N'-acetylspermidine; PUT, putrescine; SPD, spermidine; SP, spermine.

42

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

(Chun et al., 1976; Cooper et al., 1978; Rennert and Shukla, 1978), psoriasis (Cooper et al., 1978; Rennert and Shukla, 1978), and hereditary muscular dystrophies (Rudman et al., 1980). 2. In those patients with tumors with small actively growing fractions and slow growth rates, the blood polyamine contents do not always change in parallel with, or reflect the stages of, the development of the disease (Russell, 1977). 3. Plasma or serum polyamine contents, like those in urines, have been shown to decrease when antitumor therapy (either chemical or surgical) is effective, but to remain nearly unchanged with the patients are unresponsive to the chemical treatments. Furthermore, the time course of the changes in plasma or serum polyamine levels following successful therapy appears to b e roughly identical to that of the urinary contents (Marton et al., 1973b; Russell et al., 1973; Russell and Russell, 1975; Nishioka et al., 1976, 1978a; Durie et al., 1977b; Nishioka and Romsdhal, 1977, 1978; Savory et al., 1979; Hospattankar et al., 1980). 4. It must also be stressed that increases of blood polyamine concentrations can result either from an increase in their production rate or a decrease in their removal from the blood, as a consequence of failure of hepatic conjugation andlor of renal clearance. In connection with this, hepatic and renal damage are often observed in patients with advanced metastatic cancer, and serum polyamines have been demonstrated to be high in patients with uremia (Campbell et al., 1978; Swendseid et al., 1980). 5. As has been previously stated about increases in the urinary polyamine concentrations, for each of the different types of human neoplasias enhancement, when present, of polyamine levels in plasma or serum of cancerous patients is not always equally great, and we do not know which increase in polyamines is characteristic of cancer, although putrescine and spermidine are those whose levels have been shown most frequently to be augmented. Therefore, as we did for urinary polyamine levels, we have listed the papers published so far that give polyamine contents (whether increased or not) for whole blood, plasma, and serum in oncopathic humans. a. Whole Blood. Elevations of spermidine and/or spermine have been found in some patients with leukemia (Shimizu et al., 1965), gastric cancer (Saeki et al., 1978), lung cancer, non-Hodgkin’s lymphoma, and chronic lymphocytic leukemia (Cooper et al., 1978). b. Plasma. Infrequently, an elevation of plasma levels of spermidine or spermine or both has been recorded in patients with

POLYAMINES IN MAMMALIAN TULIOHS

43

laryngeal tumors (Savory et al., 1979), breast carcinoma (Chaisiri et al., 1979, 1980; Savory et al., 1979), chronic lymphocytic leukemia (Cooper et al., 1978), polycythemia Vera rubra (Desser et ul., 1975), prostatic cancer (Chaisiri et al., 1979, 1980), teratoma of the testes (Chaisiri et al., 1980), and multiple myeloma (Russell and Russell, 1975). High spermidine concentrations have occasionally been reported also in benign neoplasias of the prostate or breast, although the elevations were of a lesser magnitude than those observed in cancer patients (Chaisiri et al., 1980). In patients with either of these two types of neoplasias there were no significant differences in spermine levels between benign and malignant forms (Chaisiri et al., 1979). No correlation between elevated plasma spermidine or spermine concentrations and the tumor stage or the clinical status of the patient was possible in any kind of neoplasia. c . Serum. Total polyamines were high in some patients with melanoma (D. Bartos et al., 1975), hepatoma (D. Bartos et al., 1975), gastric carcinoma (D. Bartos et al., 1975), Wilms’s tumor (D. Bartos et al., 1975), Hodgkin’s disease (Hospattankar et al., 1980), acute myeloid leukemia (Hospattankar et al., 1980), and non-Hodgkin’s disease (Hospattankar et ul., 1980). Putrescine and spermidine were high in one patient with multiple myeloma (Russell and Russell, 1975). High spermidine was found in some patients with pancreatic carcinoma (Marton et d., 1973a,b), breast cancer (Marton et d.,1973a,b), Hodgkin’s disease (Marton et a1., 1973a,b), acute nonlymphocytic leukemia (Marton et al., 1973a,b), and acute lymphocytic leukemia (Marton et al., 1973a,b). In an interesting series of papers, Nishioka and his co-workers described the polyamine levels in patients with different types of tumors, but particularly in patients with carcinoma of the colon and the rectum. They demonstrated that patients with melanoma, Hodgkin’s disease, or carcinoma of the kidney had higher putrescine concentrations than the normal controls (Nishioka and Romsdahl, 1974; Nishioka et al., 1978a), while the spermidine level in patients with breast carcinoma was also enhanced (Nishioka and Romsdahl, 1974; Nishioka et al., 1978a). In a high percentage of patients with colorectal carcinomas, elevated concentrations of one or more polyamines have been reported (Nishioka and Romsdahl, 1974,1977,1978; Nishioka et al., 1976,1977, 1978a), while other patients with different types of benign bowel diseases had normal polyamine contents (Nishioka et ul., 1977, 1978a). Interestingly enough, careful longitudinal studies of surgical patients with colorectal carcinomas demonstrated that this is one of the few situations in which serum polyamine levels correlate fairly well, despite certain limitations, with the clinical course, especially in long-

44

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

term assessment of the disease’s activity after surgery, with or without adjunctive radiotherapy (Nishioka and Romsdahl, 1977, 1978; Nishioka et al., 1978a). Results obtained by Desser et al. (1980)are in good agreement with those of Nishioka and his co-workers. 2. Polyamine Levels in the Various Types of Formed Cells of the Blood The data available in the literature about the normal concentrations of the chief polyamines in the various types of formed cells in human blood and in human bone marrow are reported in Table V. As shown in this table, the mean concentrations of the polyamines were found to be by far higher in leukocytes than in erythrocytes (Chun et al., 1976; Cohen et al., 1976; Cooper et al., 1976, 1978; Rennert and Shukla, 1978; Saeki et al., 1978). There were no significant differences in spermidine and spermine levels among the various leukocyte types (Cooper et al., 1976, 1978; Rennert and Shukla, 1978), but polymorphonuclear cells contain more putrescine than mononuclear cells (Rennert and Shukla, 1978).Last, platelets have polyamine concentrations roughly equivalent to those of red blood cells (Cooper et al., 1976, 1978; Rennert and Shukla, 1978). This kind of quantitative distribution of polyamines cannot readily be considered to be casual, but is consistent with the fact that leukocytes are nucleated cells, whereas erythrocytes and platelets are not. In addition to quantitative differences between red blood cells and white blood cells, there is another difference in the polyamine profiles of these two cell types, since erythrocytes were found to contain more spermidine than spermine, while the opposite is true for leukocytes (see Table V) (Chun et al., 1976; Cohen et al., 1976; Cooper et al., 1978; Rennert and Shukla, 1978; Saeki et al., 1978).Furthermore, as is well known to happen in many other mammalian tissues and cells, the polyamine content in red blood cells markedly decreases with age of the cells (Cooper et al., 1976; Rennert and Shukla, 1978). When we compare the polyamine contents in the red cells and white cells of the blood not for equal numbers of cells, but in relation to the relative amounts of each of these two cell classes in the blood, since there are about 700 erythrocytes for each leukocyte in circulating blood, the most significant contribution (nearly 80%) is that from the erythrocytes, with only 20% from leukocytes (Cohen et ul., 1975, 1976; Cooper et al., 1978; Rennert and Shukla, 1978; Saeki et al., 1978). In recent years, quantitative assays of polyamines (noticeably spermidine and spermine) in erythrocytes from cancer patients have been

POLYAMINES I N MAMMALIAN TUMORS

45

suggested as a useful tool in clinical oncology. In fact, abnormally high concentrations of these two polyamines have been significantly frequently observed in erythrocytes from patients with different types of neoplasia, such as breast cancer (Savory et al., 1979; Takami et al., l979,1980a,b; Takami and Nishioka, 1980),colorectal tumors (Saeki et al., 1978; Savory et al., 1979; Takami et al., 1979,1980a7b;Takami and Nishioka, 1980; Ueharaet al., 1980a), pulmonary cancer (Cooper e t al., 1978; Savory et al., 1979; Takami et al., 1979, 1980a,b; Takami and Nishioka, 1980; Uehara et al., 1980a), melanoma (Takami et al., 1979, 1980a; Takami and Nishioka, 1980), pancreatic cancer (Saeki et al., 1978; Ueharaet al., 1980a), gastric cancer (Saekiet al., 1978; Ueharaet al., 1980a), duodenal cancer (Saeki et al., 1978), ovarian cancer (Saeki e t al., 1978), myeloblastic or lymphoblastic leukemias (Cooper et al., 1978; Savory et al., 1979), lymphoma or lymphosarcoma (Cooper et al., 1978; Saeki e t al., 1978; Uehara et al., 1980b), tumors of larynx (Savory et al., 1979)or of prostate (Savory et al., 1979), hepatoma (Saeki e t al., 1978; Uehara et al., 1980a), and reticulum cell sarcoma and Hodgkin’s disease (Uehara e t al., 1980b). No correlation was observed between leukocyte counts and increased polyamine levels in cancer patients whose leukocyte counts were within the normal range (Takami et al., 1979, 1980a). In some careful follow-up studies of cancerous patients, the concentrations of spermidine and spermine in red blood cells were markedly reduced after successful chemotherapy or after surgery in a quite satisfactory percentage of patients (Savory et al., 1979; Uehara et al., 1980a,b). Moreover, the polyamine levels in erythrocytes from patients with different types of malignant lymphoma were well correlated with the stage of the disease (Uehara et al., 1980b). However, when we consider the degree of specificity of such a new “marker” (i.e., the polyamine content in erythrocytes of oncopathic humans) for malignancy, we are once again disappointed, since increased contents of at least one or more polyamines have been found in red blood cells from patients with chronic hepatic disease (Savory et al., 1979), elliptocytosis (Cooper et al., 1978), sickle cell anemia (Chun e t al., 1976; Rennert and Shukla, 1978; Natta et al., 1980), sickle-hemoglobin C disease (Natta et al., 1980), and Duchenne muscular dystrophy (Mollica et d., 1980) and from nondialyzed patients with advanced renal failure (Swendseid et al., 1980).Nevertheless, we can answer the question as to whether determination of polyamine concentrations in red blood cells taken from cancer patients is a more sensitive and useful neoplastic “marker” than the same determination in plasma of the same patients affirmatively, since several reports have shown that the

TABLE V NORMALLEVELSOF THE CHIEF POLYAMINES IN HUMAN ERYTHROCYTES, LEUKOCYTES, PLATELETS, AND BONEMARROW Specimen and unit Erythrocytes

+dml +dml

nmol/lOBcells nm0l/l0~cells nm0l/l0~cells nm0l/l0~cells nmol/ml nmol/lO1O cells nm0l/l0~cells nmoVmg protein nmol/rng protein nmol/lO'Ocells nmol/lO" cells nmol/ml

N 4 7 9 9 17 20 27 37 11 7 7 18 22 6

Putrescine

0.02 0.007 0.07 0.01

*

0.17 0.05 0.05 1.08

*

Spermidine

* * * * * * *

1.6 0.3 1.02 0.08 1.48 1.39 0.46 0.82 0.07 1.06 0.19 14.1 3.1 15.04 3.63 11.76 ? 2.74 0.55 2 0.55 3.21 5 1.82 14.42 3.2 15.58 ? 3.9 24.8 2 6.3

*

Spermine

Remarks

Reference

0.2 0.89 2 0.28 0.61 0.9 0.27 0.48 0.04 0.46 0.09 8.4 2 2.8 8.8 3.12 7.21 2.29 1.13 2 0.54 0.53 2 0.75 8.72 2.7 8.85 2 2.8 12.4 3.4

SD

Shimizu et al. (1965) Cohen et al. (1976) Cooper et al. (1976) Chun et al. (1976) Cooper et al. (1978) Rennert and Shukla (1978) Saeki et al. (1978) Uehara et al. (1980a) Takami and Nishioka (1980) Natta et al. (1980) Natta et al. (1980) Uehara et al. (198Ob) Uehara et al. (198Ob) Swendseid et al. (1980)

* * * * * * *

SD SD SD Stm LY s SD, M SD, W

M, SD

Leukocytes nmol/108 cells nmol/ 10’ cells nrnol/1O9 cells nmolll0’ cells nm01/10~cells nmol/109 cells nmol/io9cells nmoUl0’ cells nmol/lO9 cells nmol/lO’ cells Platelets nmol/lO’ cells nmol/lO’ cells nmolll0’ cells Bone marrow nmol/ml

* 1.4 2.99 * 1.5

* * * * * *

3.0 2 0.9 126 t 31 15.3 0.5 253.6 94.9 241.3 155.5 95 26 226 28 266.5 t 28.2 207.3 45.5 207 45

* * * * * *

12.9 3.8 357 t 105 35.95 0.91 490.2 208.7 547.6 183.4 387 5 61 440 61 440.3 2 61.4 493.1 56.3 493 2 56

L SD, M N SD, PMN SD MN MN PM N PMN

Desser et al. (1975) Cohen et al. (1976) Chun et al. (1976) Cooper et al. (1976) Cooper et al. (1976) Saeki et al. (1978) Cooper et al. (1978) Rennert and Shukla (1978) Rennert and Shukla (1978) Cooper et al. (1978)

0.11 0.05 0.21 0.03 0.11 * 0.05

0.12 5 0.06 0.44 0.05 0.12 * 0.02

*

0.04 2 0.03 <0.043 0.04 0.01

SD

Cooper et al. (1976) Cooper et al. (1978) Rennert and Shukla (1978)

2.04

27.9

31.6

1.8

3 4 9 10 10 7 17 20 20 17

140 60.6 467.5 2 244.3 220 50 140 60.6 467.5 244.3 410 2 80

6 17 20 5

-

* * *

* *

*

*

Miale et al. (1977)

*

“The results reported are expressed as means alone, or means SE, or means SD (when specified). N , number of subjects; L, lymphocytes; MN, mononucleated leukocytes; PMN, polymorphonucleated leukocytes; Lys, lysate; Stm, stroma; M, men; W, women.

48

GIUSEPPE SCALABRINO AND MARIA E . FERIOLI

percentage of oncopathic subjects with elevated polyamine levels in their erythrocytes is far higher than that of patients with elevated plasma polyamine levels (Savory et al., 1979; Takami et al., 1979, 1980a,b). White blood cell preparations from patients with chronic myelocytic leukemia contain more SAM than normal peripheral white cells or thoracic duct lymphocytes (Baldessarini and Carbone, 1965).

3. Polyamine Levels in Bone Marrow There have been very few studies concerning this particular aspect, carried out mainly with bone marrow aspirates from children with different forms of leukemia, and a few cases with other malignancies or other hyperproliferative hematologic nonmalignant disorders, such as infectious mononucleosis, sickle cell anemia, and histiocytosis X have been included (Rennert et al., 1976b; Miale et al., 1977). Even fewer are the data available on the polyamine contents in normal bone marrow from control subjects (Miale et al., 1977). What is available is reported in Table V. In various nonneoplastic diseases, very low levels of putrescine, even lower than normal values, have been found (Rennert et al., 1976a; Miale et al., 1977). However, surprisingly enough, very elevated contents of all the polyamines were detected in bone marrow taken from patients with hyperproliferative nonneoplastic hematologic diseases, such as infectious mononucleosis, or with nonhematologic neoplasias, such as retinoblastoma (Rennert et al., 1976b; Miale et al., 1977). Marked elevations of the concentrations of putrescine, spermidine, and spermine in a high percentage of patients either with acute lymphocytic or with acute myelocytic leukemias in relapse were observed (Nishioka et al., 1976; Rennert et al., 1976b; Miale et aZ., 1977). On the contrary, remission in the leukemic patients, regardless of the type of leukemia, was accompanied by an impressive fall in the polyamine concentrations (Nishioka et al., 1976; Rennert et al., 1976b; Miale et al., 1977). Very noteworthy is the observation of Miale et al. (1977) that in some patients there was a much more prominent increase in putrescine level than in spermidine and spermine that preceded by several weeks detection of fulminating systemic relapse of the leukemia. Therefore, in the bone marrow as well, monitoring of the putrescine content is a good biochemical indicator of the clinical status of a leukemia patient (Miale et al., 1977). However, in conclusion, from the few data available in the literature it is apparent that increased polyamine levels in bone marrow cannot be considered to be pathognomonic for neoplastic disorders, whether hematologic or not.

POLYAMINES IN M A M M A L I A N TUMORS

49

The polyamine levels in bone-marrow plasma obtained from patients with different types of leukemias have been measured (Nishioka et al., 1980). Untreated patients with chronic leukemias showed higher polyamine levels than the untreated patients with acute leukemias (Nishioka et al., 1980). Moreover, patients who were in remission showed low polyamine levels, whereas patients who responded to chemotherapy showed high polyamine levels, as a consequence of the release of polyamines from tumor cells killed by chemotherapy (Nishioka et al., 1980). Finally, the topic of polyamines in blood, bone marrow, and other physiological fluids as possible “markers” of malignancy for human leukemias and other hematologic tumors has been reviewed by Desser (1980).

D. LEVELSOF THE CHIEF POLYAMINES I N PHYSIOLOGICAL FLUIDS OTHER THANBLOODAND URINE Polyamines from neoplastic cells could be released into human extracellular fluids other than blood and urines, causing elevated concentrations of these compounds. These other physiological fluids are the cerebrospinal fluid, bile, duodenal fluid, sweat, saliva, and amniotic fluid. The studies available so far on normal polyamine levels in these body fluids are very few, and all the data available on this topic are summarized in Table VI. There have been no studies of changes in polyamine contents in these physiological fluids from patients with tumors of the organ or tissue from which the liquid arises or in which the liquid is retained, or which are immersed in the liquid, with the noticeable exception of cerebrospinal fluid. Polyamine patterns in the cerebrospinal fluid of patients with tumors of the central nervous system (CNS) or tumor not arising from the CNS but involving it with meningeal carcinomatosis have been determined. In patients with meningeal carcinomatosis from breast or lung or colon or bladder cancers, marked elevations of putrescine and spermidine values were observed in comparison with the reference group, which consisted of subjects with non-CNS tumors, but tumors with no evidence of CNS involvement (Yap et al., 1979). Additionally, in children with acute lymphocytic leukemia with or without involvement of the CNS, the free polyamine concentrations of the cerebrospinal fluid were measured (Rennert et al., 1977). Significant increases of spermidine and spermine, but not of putrescine, were found in patients with florid CNS leukemia in relapse, as compared with patients with extramedullar leukemia but without cytologic evidence of CNS in-

NORMAL LEVELS

OF THE

Specimen

Unit

Cerebrospinal fluid Bile Duodenal fluid Sweat Saliva Amniotic fluid

pmoUml Ccdml Ccg/ml Ccg/ml Ccdml pglrng CR

CHIEF POLYAMINES

N 5 2 5 4 4 230

IN

TABLE VI HUMANPHYSIOLOGICAL

FLUIDS

OTHER THANBLOODAND URINE"

Putrescine

Spermidine

Spermine

Remarks

Reference

182 2 79 -

1 2 0 2 34 14.6 1.9 5 0.3 C0.25 <0.05 0.33 2 0.1

ND 16.1 1.27 2 0.6 c0.25 (0.05 0.76 0.2

SD SD -

Marton (1978); Marton et al. (1979) McEvoy and Hartley (1975) McEvoy and Hartley (1975) McEvoy and Hartley (1975) McEvoy and Hartley (1975) Russell et al. (1978)

-

-

0.96

* 0.2

*

-

-

a The results reported are expressed as means alone, or means 2 SE, or means 2 SD (when specified). ND, not detectable; N, number of subjects; CR, creatinine.

POLYAMIKES IN MAMMALIAN Tl1MORS

51

volvement (Rennert et al., 1977). Strikingly enough, leukemia patients with extramedullary localization, but without cytologic and syniptomatic evidence of CNS involvement, had the highest levels of putrescine of all the groups of patients (Rennert et al., 1977). Certainly, assays for polyamines in the cerebrospinal fluid are of particular clinical importance when they are connected with pathologies of the CNS, whether neoplastic or not. Without any doubt, we owe a debt to Marton and his co-workers, who studied very carefully and thoroughly the derangements of polyamine profiles in the cerebrospinal fluids of patients with neoplastic and nonneoplastic disorders and have tried to establish some connections between these abnormalities and the clinical status of the patient. In a series of patients with different types of CNS tumors, such as glioblastonia, medulloblastoma, astrocytoma, pituitary adenoma, meningioma, ependymoma, and acoustic neuroma, Marton and his co-workers (1974a,b, 1976; Marton, 1977) found elevated putrescine and spennidine concentrations in cerebrospinal fluid of most cases, particularly those with glioblastoma and medulloblastoma, whereas patients with astrocytoma had less consistent increase in these polyamines. But all the patients with any one of these three types of tumor always had polyamine levels higher than those found in either normal controls or the humans with various nonneoplastic disorders of the CNS (namely, infectious diseases, demyelinating diseases, stroke) and used as the reference group (Marton et al., 1974a,b, 1976; Marton, 1977, 1978). Meningiomas are a variable group of tumors, without any characteristic pattern of polyamine elevation in cerebrospinal fluid (Marton et al., 1976; Marton, 1977). Elevation of spermine levels in the spinal fluids of tumor patients was sporadic (Marton et al., 1976; Marton, 1977). Furthermore, patients who underwent successful therapy showed an immediate rise in putrescine concentration, followed later by a marked decrease in polyamine content (Marton et al., 1974a, 1976; Marton, 1977,1978). In the assessment of the disease’s activity in several patients with CNS tumors, who exhibited significant new increases in putrescine concentration, it was observed that recurrence of the neoplastic disease and a decline in the clinical state of the patients followed (Marton et al., 1976; Marton, 1977, 1978). It is of interest that the fluctuations in the concentrations of polyamines in cerebrospinal fluid from patients with CNS tumors do not have any relationship to changes in protein concentration in the same liquid, suggesting that these changes are not merely mirroring some alteration in the bloodbrain barrier (Marton et al., 1976). Polyamine assays of cerebrospinal fluids from CNS cancer patients

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have been demonstrated to be helpful in short-term evaluation of the efficacy of a specific course of therapy. They may also, at least in some particular types of CNS neoplasias, be h e l p h l for long-term evaluation of tumor relapse or regression. This is true for medulloblastoma patients, where Marton et d. (1979)demonstrated an absolute correlation between polyamine (particularly putrescine) levels and the clinical status, evaluated b y different radiographic techniques and by cytological criteria. In fact, almost all the patients exhibited appropriate decreases in polyamine values in response to chemotherapy or radiotherapy, and 50% of the same patients showed appropriate increases in polyamine levels several weeks before the recurrence of the disease. This is of great interest, since medulloblastomas, because of their localization, pose particular difficulties for assessment of their activity and progress, with radiographic techniques frequently of little use (Marton et al., 1979). On the contrary, in patients with glioblastoma multiforme or anaplastic astrocytoma, it has been shown that cerebrospinal fluid polyamine level determinations were not as helpful as in patients with medulloblastoma for monitoring tumor progression and for forseeing tumor recurrence (Fulton et aZ., 1980). In fact, the putrescine and spermidine levels in all the patients with glioblastoma multiforme or anaplastic astrocytoma were significantly higher than those of the reference group of patients with nonneoplastic CNS disorders, but there was no difference in polyamine levels between the two groups of tumor patients, in spite of the fact that the degree of malignancy and the fraction of proliferating cells in glioblastoma multiforme are higher than in anaplastic astrocytoma (Fulton et al., 1980). Moreover, no significant relation was found between the enlargmenet of the tumor and the polyamine levels in cerebrospinal fluids of patients with these two kinds of CNS neoplasia (Fulton et d.,1980). The striking discrepancy in the correlation of cerebrospinal fluid polyamine levels with tumor relapse between medulloblastoma and malignant supratentorial gliomas may be connected with the fact that polyamines produced by malignant hemispheric gliomas cannot reach the cerebrospinal fluid or reach it with difficulty (Fulton et al., 1980). In fact, malignant supratentorial gliomas are distant from the cerebrospinal fluid pathways, whereas medulloblastomas are generally located adjacent to the cerebrospinal fluid pathways (Fulton et al., 1980). On the whole, we can conclude that increases in polyamine contents in whatsoever physiological fluid or in more of the physiological fluids of beings ill with cancer (regardless of the type of cancer), when present, cannot be considered to be biochemical “markers” specific for tumor cell proliferation, but rather are merely biochemical “markers”

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of cell growth. In other words, the increases in intra- and extracellular polyamine levels may reflect cell multiplication, whether the type of growth is controlled or uncontrolled.

E. LEVELSO F ACTIVITYOF POLYAMINE BIOSYNTHETIC DECARBOXYLASES I N HUMANNEOPLASTICTISSUES I N RELATIONT O THE DEGREE O F MALIGNANCY Because of all the doubts about the significance of polyamine determinations in biological materials from cancers and because of the futile question as to which polyamine it would be the best to measure in each of the various types of human neoplasia, we decided to measure the levels of polyamine biosynthetic decarboxylase activities in different types of primary human neoplastic tissues. In fact, in experimental oncology research with Morris rat hepatomas with vastly different growth rates (Williams-Ashman et aZ., 1972), and with chemical carcinogenesis in mouse skin (O’Brien, 1976; Boutwell et al., 1979), it has been found that the degree of ODC enhancement can be a useful biochemical indicator of neoplastic growth, with some few exceptions for certain kinds of rat hepatomas (Pariza et aZ., 1976). We chose human tumors that could be easily obtained from the operating room or from circulating blood. Accordingly, we studied cutaneous epitheliomas, brain tumors, and leukemias. In the cutaneous epitheliomas, the assays of the enzymes were carried out only in the neoplastic epidermal layer, after its separation from the dermis (Scalabrino et al., 1980). Metastases of tumors to the brain or to the skin were deliberately excluded from our research, because of their mixtures of normal cells and neoplastic cells, which, coming from the organ site of the primary tumor, have a different histogenetic origin. Furthermore, it is well known that the cell type and cell viability are often different in metastases than in primary tumor cells. Our major finding is the difference in levels of activity of polyamine biosynthetic decarboxylases in various tumor types. The degree of enhancement of ODC activity correlates well with the neoplasm’s growth rate, since among cutaneous epitheliomas it is greater in squamous cell carcinomas than in basal cell epitheliomas (Scalabrino et al., 1980).It is well established, in fact, that basal cell epithelioma is a slow-growing tumor, whereas the squamous cell carcinoma has a faster growth rate and more widespread local invasiveness. Again, the magnitude of the elevation of ODC activity in the CNSrelated tumors was also proportional to the malignancy. In fact, the levels of ODC activity are higher in the group of dedifferentiated

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astrocytomas (i.e., astrocytomas of grades 3 and 4,astroblastoma, and glioblastoma multiforme) than in the group of differentiated astrocytomas (i.e., astrocytomas of grades 1 and 2 ) (Scalabrino et al., 1981).The same is true for the meningiomas, among which the typical forms have lower ODC activity than the atypical forms (Scalabrino et al., 1981).Polar spongioblastoma, which is generally held to be a quite slowly growing intracranial tumor, has the lowest ODC activity among the brain tumors we have tested. Medulloblastomas, highly malignant brain tumors, show the highest ODC activity of all the other tumors of the CNS tested (Scalabrino et al., 1981). Our data are fairly in agreement with the data of Harik and Sutton (1979), who found a higher content of putrescine in CNS tumors in relation with increasing malignancy. However, although the number of patients screened by Harik and Sutton (1979) is undoubtedly larger than ours, medulloblastomas were not tested and no differentiation within the meningiomas was made in that report. We also reported a dramatic fall in ODC activity in leukocytes taken from patients with chronic myeloid leukemia after successful chemotherapy (Scalabrino et al., 1981). When we considered instead the course of the changes in SAMD activity in the same different types of tumors, we saw that the degree of enhancement of this enzymic activity also followed well the degree of malignancy of the tumor. The only exception to this general statement is the surprisingly low activity of the enzyme in the medulloblastomas we have studied. We have no explanation for this finding. Moreover, unlike ODC activity, SAMD activity does not reflect the effectiveness of antineoplastic chemotherapy, since its level in leukemic leukocytes is not changed immediately after treatment (Scalabrino et al., 1981). At present it is quite difficult to interpret this discrepancy in the behaviors of the levels of the two polyamine biosynthetic decarboxylases in response to the same type of therapy. In conclusion, our data, although limited in number for certain types of tumors, prove the clever forecast of Bachrach (1976b), who asserted that “it is not unlikely that the activity of this enzyme (i.e., ODC) in biopsy material may aid the pathologist in the diagnosis of malignancy.” In fact, we have shown that the degree of ODC enhancement correlates well with the degree of malignancy in different types of human tumor, even those with different histogenetic origins. However, we cannot consider the elevation in ODC activity in neoplasias to be specific for the processes of neoplastic transformation and growth, since it has been demonstrated that similar elevations occur with many other processes of nonneoplastic growth, i.e., of controlled growth

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(Janne et al., 1978; Russell and Durie, 1978). Rather, on the basis of our findings, we consider the degree of elevation in ODC activity observed in human tumors to be clinically useful as an indicator of a neoplasm’s growth rate, which means of the degree of malignancy of the tumor. This correlation also agrees with idea of Helson et al. (1976, 1977), based on their findings in cultured human neuroblastoma cells (1976) and human melanoma tumors grown in Swiss nulnu mice

(1977).

In certain experimental tumors, such as Morris hepatomas (Williams-Ashman et al., 1972) and epithelial tumors of mouse skin (O’Brien, 1976; Boutwell et al., 1979),a dichotomy between ODC and SAMD activity has been demonstrated, with SAMD levels increased not at all or only a little. On the contrary, in the human tumors we tested, SAMD activity increases in parallel with, although to a lesser extent than, the increase in ODC activity. However, the idea that SAMD activity levels also mirror neoplastic growth rates must be taken with caution, because of the exception of the medulloblastoma and, theoretically, of other human tumors not yet assayed. Last, when we compare the reliability of determinations of polyamine contents with that of determinations of the levels of the polyamine biosynthetic decarboxylases as diagnostic and chiefly prognostic tools in different areas of clinical oncology, our conclusions are that (a) measurements of polyamine levels in physiological fluids can be useful for short-time evaluation of a specific course of therapy and for detection of remission or relapse of the neoplastic disease, but are of little or no use in evaluating the degree of malignancy of the tumor, even when combined with assays of other tumor “markers” in neoplastic tissue or in physiological fluids from cancer patients; (b) the levels of the polyamine biosynthetic decarboxylases are by far the better indicators of the degree of malignancy of the tumor, but of no use in evaluating the effectiveness of therapy, except for hematologic neoplasias, in which the assays of enzyme levels in tumor cells are obviously repeatable.

F. METABOLIC CONJUGATION O F POLYAhlINES AND ANTIPOLYAMINE ANTIBODIES I N NORMALSA N D I N CANCER PATIENTS The polyamine levels in physiological fluids are clearly sustained by regulatory interplay of several pathways: (a)de nouo synthesis; (b) release from the intracellular compartment; (c) metabolic transformation into conjugated forms; (d) catabolic transformation into degradation products; (e)excretion. Among these pathways, the in uivo metab-

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olism of injected labeled putrescine and labeled spermidine have been studied in normal volunteers (Rosenblum et al., 1977, 1978a,b; Rosenblum, 1980) as well as in cancer patients with high endogenous plasma levels of polyamines (Rosenblum et al., 1977, 197813; Rosenblum, 1980). These studies demonstrated that the radiolabeled compounds very rapidly disappear from the plasma of both normal subjects and cancer patients, with mean half-life times that were very short and very similar for the two groups. This was because of the quick transformation of the labeled polyamines into their conjugated forms, which in turn have prolonged half-life times in plasma. Accordingly, most of the injected radioactive polyamines was excreted in the days after injection almost exclusively in conjugated form (Rosenblum et al., 1977,197813; Rosenblum, 1980). All these results suggest that in neoplastic patients, unlike in patients with cystic fibrosis (Rosenblum et al., 1978a; Prussak and Russell, 1980), the metabolic transformation capacity is not impaired, in spite of the fact that extensive production and marked release of polyamines into physiological fluids by neoplastic cells frequently occurs. Therefore, one can conclude that the conjugating pathways for polyamines have high functional capacity and a high saturation level (Rosenblum, 1980). As for natural antibodies to polyamines in sera of normal or cancer patients, recent preliminary results from Roch et al. (1979) suggest that the titers of such antibodies may be quantitatively lower in the sera of tumor patients than in the sera of normal controls. It is desirable to expand this kind of research, to obtain more data, and to clarify the pathophysiological significance of such immunological differences. Ill. Diamine Oxidase Activity in Human and in Experimental Neoplasms

One explanation for the elevated putrescine concentrations observed during neoplastic growth is the increased activity of ODC (see Sections I1 and 111, Part I, Vol. 35). But another enzyme that must also play a role in determining its concentration is diamine oxidase [amine : oxygen oxidoreductase (deaminating) (pyridoxal containing); E C 1.4.3.61 (DAO), which converts putrescine into y-aminobutyraldehyde (see Section I,E, Part I, Vol. 35). Diamine oxidase has been proved to be the same as histaminase, as this enzyme was called several years ago. Because histamine is not the only substrate for this enzyme, which also catalyzes the deamination of putrescine and of several polyamines, including cadaverine, diaminopropane, and a series of aliphatic diamines, the more inclusive term “diamine oxidase” has been proposed. We will use the term “diamine

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oxidase” to indicate this enzyme cven when the authors reviewed used the old term “histaminase.” A. IN HUMANTUMORS Increased DAO activity has been shown to b e associated with several types of human cancer. In a series of papers, Baylin and his co-workers (1970, 1972a,b) provided evidence that some patients with medullary carcinoma of the thyroid had abnormally high DAO activity in both the serum and tumor specimens. Although the tissue samples were obtained at autopsy and the controls, instead of being normal healthy subject, had died from other neoplastic diseases (breast carcinoma, osteosarcoma, Ewing’s sarcoma), Baylin and his co-workers should still be considered the pioneer workers in this field. After these first results showing high DAO activity in serum of patients with medullary carcinoma of the thyroid, Baylin and his co-workers and other authors continued the studies of this tumor and compared the usefulness of measurement of DAO activity with that of measurements of calcitonin levels to see whether also the measurement of the activity of the enzyme in serum of patients might be useful for early detection of localized tumor (Baylin et al., 1970) as well as for metastases and residual tumor after surgery (Baylin et al., 1970, 1972a,b; Keiser e t al., 1973). After removal of tumor, the serum DAO activity in patients with localized tumor declined to normal, but it remained high in many patients with metastatic tumor (Keiser et al., 1973). This confirms the previously reported correlation between high serum DAO activity and metastatic disease (Baylin et al., 1972a). However, unlike calcitonin, serum DAO levels are not elevated in all patients with medullary carcinoma of the thyroid, and for this reason measurements of serum-calcitonin levels seem to be a more reliable test for the diagnosis of this neoplasm (Baylin et d., 1972a; Keiser et al., 1973). In a more recent paper, it was confirmed that high plasma DAO levels are not characteristic of patients with occult thyroid tumors or C-cell hyperplasia (Mendelsohn et al., 1978). On the contrary, there is DAO within the tumor cells, and it is almost certainly produced by them (Mendelsohn et al., 1978). In addition, DAO has been shown to be present only in certain cells of medullary carcinoma of the thyroid, but not in normal or hyperplastic C cells (Mendelsohn et al., 1973). Earlier studies also showed that this thyroid tumor contained many times more DAO activity than the normal adjacent thyroid tissue, suggesting that the large amounts of DAO in serum had originated from

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the tumor (Baylin et al., 1970).In fact, the reappearance of DAO activity in serum after the administration of aminoguanidine (a known specific inhibitor of DAO) coincides with new synthesis of the enzyme in the tumor (Baylin et al., 1970, 197213). This has been proved by administration of aminoguanidine to patients with high serum levels of the enzyme: the enzyme disappeared and then reappeared over a period of days at a rate that indicated total turnover of the enzyme in the serum in about 2 days (Baylin et al., 1970). In other words, the tumor continuously produced and released the enzyme into the circulation. This release was not affected by heparin, which does promote the entry of DAO from normal tissues into the circulation (Ettinger et al., 1978). Elevated DAO activity in effusion fluids was reported by Lin et al. (1975), associated with a number of other human cancers, including those of the ovary, breast, stomach, colon, and lung. These authors found an increase in the enzyme activity in ascites fluids from cancer of endometrium, stomach, colon, and in pleural fluids obtained from subjects with cancer of the lung and breast. An elevation of DAO activity was also found to concur with the presence of the Regan isoenzyme of alkaline phosphatase, which is known to be associated with a number of human tumors (Lin et al., 1975). The results of Lin et al. (1975),and other earlier results showing high DAO activity in plasma of patients with endometrial adenocarcinoma, uterine myosarcoma, and granulosa cell carcinoma (Borglin and Willert, 1962), were further evidence that the idea of Baylin et al. (1970, 1972a,b) that increased DAO activity in serum is a specific marker for medullary thyroid carcinoma is not correct. However, Baylin and his co-workers and other authors proposed that measurement of serum DAO activity might be diagnostic for this kind of tumor if combined with simultaneous measurements of serum calcitonin levels (Baylin et d., 1972a; Keiser et d., 1973; Mendelsohn et al., 1978). Lin et al. (1979)extended their studies to evaluation of DAO activity in over 400 malignant effusion fluids (pleural, peritoneal, or pericardial) collected from 162 cancer patients. Elevated DAO activity was found in a larger percentage of the effusion fluids of patients with cancers of the ovary, colon, and stomach (Lin et al., 1979).In patients with cancer of the colon and stomach, the elevation of DAO was found to be correlated with the production of carcinoembryonic antigens in the majority of the cases; whereas among those with cancer of the ovary, the elevation of DAO tended to go along with the production of the &subunit of human chorionic gonadotropin (Lin et al., 1979). Results of Ettinger et a2. (1980) confirmed those of Lin et al. (1979)and

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demonstrated that the ovarian carcinoma cell appears to be the source of the increased DAO activity in the ascitic fluid from some of these patients. Furthermore, in this type of oncopathic subject, DAO activity is consistently greater in ascitic fluid than in plasma (Ettinger et al.,

1980). The results of Kusche et al. (1980) contrast with the results above. They reported that the distribution of DAO activity in the gastrointestinal tract of patients with adenocarcinoma of the large bowel or of the stomach did not indicate that the enzyme was all produced by tumor

cells. There was less enzymic activity in the tumor tissue itself than in the adjacent, histologically normal mucosa (Kusche et al., 1980). Nevertheless, the DAO activity of the gastrointestinal mucosa adjacent to tumor was influenced by tumor growth (Kusche et al., 1980). Because of its supposed origin from the neural crest and its embryological relationship to medullary thyroid carcinoma, another tumor whose DAO activity was well studied is small-cell carcinoma of the lung. This tumor was also found to have higher DAO activity than normal lung (Baylin et al., 1975). This activity has been found to be frequently elevated also in plasma of patients with small-cell carcinoma of the lung (Baylin et d.,1975). Further studies confirmed that the increase in plasma DAO activity only partially reflects the increased DAO activity in neoplastic tissue of patients with this kind of tumor, since despite the high DAO activity in the neoplastic tissue, the majority of these oncopathic subjects did not have elevated blood levels of DAO activity (Ettinger et al., 1978). Consequently, the preceding idea that the monitoring of DAO in plasma of patients with small-cell carcinoma of the lung and elevated plasma levels might have prognostic importance, indicating the state of differentiation of the tumor tissue (Baylin et al., 1975), should be discarded. In addition, a specific association of the elevation of DAO activity in the effusion fluids of patients with small-cell carcinoma of the lung was not observed in a more recent study (Lin et nl., 1979). On the other hand, Baylin et al. (1978) reported that the circulating levels of DAO, like the levels of such other markers as L-DOPA decarboxylase and calcitonin, cannot necessarily be expected to mirror tumor growth in patients with small-cell carcinoma of lung. It is possible that the variable biochemical patterns in different tumors from different patients and even in a single neoplasm may reflect heterogeneity of cell population and metabolic activities in the neoplastic tissue, because small-cell carcinoma can originate in more than one clone of cells, each with different patterns of biochemical expression and storage content of markers (Baylin et d., 1978).

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In a study to ascertain whether small-cell carcinoma of lung might have a separate histogenesis from the other major types of human lung tumors, it was shown that the differences in DAO activity in the major forms of human lung cancer are quantitative rather than qualitative (Baylin et al., 1980a). The occurrence of increased DAO activity is roughly the same in the various histopathological types of lung neoplasms in humans (Baylin et al., 1980a). In contrast to L-DOPA decarboxylase activity, which appears to be a valuable marker for differentiating small-cell carcinoma cells from other lung cancer cells in uitro, DAO activity is generally low in all types of lung carcinoma cells in culture (Baylin et al., 1980a). Although the factors responsible for the increased DAO activity in tumors have not been fully delineated, it has been proposed that DAO activity in placental and neoplastic tissues is an expression of a mature genome, but not a unique expression of a fetal genome (Baylin, 1977). Although human placental histaminase is identical with histaminase of the medullary thyroid carcinoma in several biochemical features, the placental enzyme also has similarities with the histaminases of human kidney and of human intestine (Baylin, 1977). This is consistent with earlier suggestions that placental DAO is associated not with trophoblastic tissue, but with maternal decidual elements in the placenta (Lin et al., 1975). The study of Baylin (1977) also emphasizes that high DAO activity in human neoplasms might or might not be considered as an “ectopic protein production.” Last, in addition to those mentioned above, the findings about DAO activity are widely variable for other types of human cancer. In one patient with choriocarcinoma not associated with mole, an initial highly elevated serum level of DAO, comparable to the elevation found in late normal pregnancy when values are at the peak (Torok et al., 1970), was found. One patient with adenocarcinoma of the breast also had enzyme activity similar to that in a normal 8-ll-week gestation (Torok et al., 1970). There was no enzyme activity in three patients with teratocarcinoma of the testes (Torok e t al., 1970). TUMORS B. I N EXPERIMENTAL Studies by Quash and his co-workers demonstrate that there are variations in the activities of DAO and polyamine oxidase (PAO) associated with the growth of the normal and transformed cells. As cells approach confluence, normal cells have about twice the DAO activity of their transformed counterparts. This has been reported for rat kidney cells, both normal and transformed by avian sarcoma virus (Quash

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et al., 1979). Normal and virus-transformed BHK cells grown in cultures have differences in asparagine decarboxylation, which is increased in BHK cells transformed by either hamster sarcoma virus or polyoma virus (Quash et al., 1976). Asparagine not only stimulates DAO activity (Quash et al., 1976), but has been demonstrated to increase the activity of ODC in cultured neuroblastoma cells (Chen and Canellakis, 1977) (see Section 1,A). Asparagine, indeed, may exert two crucial and opposite effects on cellular polyamine metabolism, namely, stimulation of synthesis by activating ODC and enhancement of degradation by activating DAO. Because a decrease of DAO activity similar to that reported for transformed cells has also been observed in rat mammary tumor induced chemically with 9,10-dimethylbenz[a]anthracene, in leukemic myeloblasts and in a human epithelioid carcinoma cell line, the differences in DAO activity do not seem to be linked to viral transformation only, but rather to the neoplastic state (Quash et al., 1979). However, a much larger systematic study with different types of tumor will be necessary before we can conclude that diminished DAO activity is a characteristic of all types of transformed cells. However, an exactly opposite finding has been reported with an astounding elevation of DAO activity in 4-DAB hepatoma and in Yoshida ascites hepatoma cells (Perin et al., 1979). A study b y Bachrach (1980a) demonstrated that the addition of complete serum-containing medium to confluent cultures of glioma cells increased not only the activities of the polyamine biosynthetic decarboxylases, but also that of DAO. This last enzyme increase was maximal at 8 hr, when the ODC activity also reached its peak, and was followed b y the accumulation of y-aminobutyric acid, which was detected in the cells and in the medium (Bachrach, 1980a). Asparagine caused an increase in DAO activity of glioma cells in culture and enhanced the formation of y-aminobutyric acid from putrescine (Bachrach, 1980a). This can be explained by the activation of DAO by 2-oxosuccinamate, which is an intermediate of aspargine decarboxylation (Quash et al., 1976, 1979) (see below). Regarding its intracellular regulation, DAO activity can be affected i n uitro by metabolites of naturally occurring amino acids: 2-0x0succinamate, which is derived from asparagine by transamination, was found to b e an activator; oxaloacetate, which can be formed from aspartate by transamination or from 2-oxosuccinamate by enzymic deamination, was found to be an inhibitor, as is pyruvate, formed by decarboxylation of oxaloacetate (Quash et al., 1976). The activation of DAO b y 2-oxosuccinamate seems to be of limited physio-

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logical significance, because a relative high concentration is needed to produce a comparatively small activation (30-50%) of putrescine oxidation (Quash et al., 1976). Its real physiological role may be as an allosteric effector of the enzyme for substrates other than putrescine (Quashet al., 1976). On the other hand, the DAO inhibition could have physiological significance. In fact, almost total inhibition (94%)occurs with pyruvate (Quash et al., 1976), and it is well known that an elevated pyruvate concentration results from an increased rate of glycolysis, which is exhibited frequently by some types of malignant cells. IV. Physiological and Pharmacological Inhibitors of Polyamine Biosynthesis in Neoplastic Tissues or Cells

In these last years, some physiological and/or pharmacological inhibitors of polyamine biosynthesis have been used to investigate the metabolic consequence of depriving normal or neoplastic cells of polyamines. We believe that this is only one way, and a rather marginal one, to elucidate the role of polyamines in the different types of cellular growth process, both controlled and uncontrolled. Moreover, in our opinion, it would be more useful to attempt to clarify the functions of polyamines using physiological inhibitors rather than to further screening for new molecules able to inhibit polyamine synthesis. As has been reported, the depletion of polyamines in neoplastic cultured cells b y induction of ODC antizyme production provides an alternative to the pharmacological competitive inhibitors for investigating polyamine-dependent metabolic pathways in cells (Branca and Herbst, 1980). Ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase are the most suitable target enzymes for inhibition of polyamine biosynthesis because they are rate-controlling enzymes in the pathway. Substances that can control the enzyme synthesis of ODC and SAMD can be categorized first by whether they occur physiologically. Among the physiological substances, polyamines, especially putrescine, play important roles. On the other hand, most of the pharmacological inhibitors now available and used in attempts to block the accumulation of polyamines in vivo or in vitro are inhibitors of these two decarboxylases. We will consider the physiological inhibitors and the pharmacological ones separately. The reader can obtain more detailed information about the different inhibitors of the polyamine biosynthesis from the excellent reviews provided by Williams-Ashman et al. (1976),Janne e t al. (1978), Mamont et al. (1980), and Stevens and Stevens (1980).

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PHYSIOLOGICAL INHIBITORS AND

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RELATED COMPOUNDS

The naturally occurring polyamines, putrescine, spermidine, and spermine, acting as negative regulators of their biosynthetic decarboxylases, can inhibit cell growth b y themselves or through their oxidized products. We will consider both these aspects, and, because the oldest reports are devoted to the cytotoxic or antiproliferative properties of polyamines, we shall begin by reviewing the inhibitory effects of polyamines on various types of tumors, both in vivo and in vitro. Boyland (1941) found that cadaverine and spermine were effective in inhibiting the growth of grafted sarcomata and spontaneous carcinomata in mice. He demonstrated that these aliphatic bases were the factors responsible for the inhibition of tumor growth by a muscle extract. Subsequently, the effects of biogenic polyamines, including cadaverine, on the growth of cancer cells were examined in vitro in Ehrlich solid tumor and Yoshida sarcoma. None of the amines examined had any effect on the Ehrlich solid tumor, while all the amines were inhibitory for Yoshida sarcoma, spermine having the strongest effect (Miyaki et al., 1960). This inhibitory effect on cell growth is a property of polyamines in the presence of calf serum, in which situation they have a potent cytotoxic effect on tissue culture cells. It must be calf serum, since no such effect is observed with horse or human serum (Alarcon et al., 1961; Alarcon, 1964). The interpretation has been that polyamines are not cytotoxic by themselves, but become so through the action of polyamine oxidase, which is present in calf serum but not in human or horse serum (Hirsch, 1953a,b; Taboret al., 1954). In the absence of amine oxidase, the polyamines did not affect the multiplication of the Ehrlich ascites cells, whereas the toxicity of purified oxidized spermine for cells of this type has been confirmed in an in vitro-in vivo system (Bachrach e t al., 1967). Allen et al. (1979) further confirmed that the inhibition of growth is due to a bovine plasma oxidase that converts the polyamines to the inhibitory factors. They found that spermidine and spermine are potent in vitro inhibitors of proliferation of phytohemagglutinin-stimulated lymphoma cells and human lymphoblastic leukemia cells only in media supplemented with fetal calf serum. In addition, putrescine, which was not an inhibitor in the presence of fetal calf serum, become so in the presence of human pregnancy serum, possibly due to its content of DAO (Allen e t al., 1979). Diamine oxidase degrades its substrates to aldehydes, which in general are cytotoxic, and it is probably in this way that DAO inhibits proliferation of Bri8 human leukemic lymphocytes (Gaugas, 1980). This is supported by the fact that amino-

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guanidine can reverse the DAO-induced inhibition (Gaugas, 1980). However, the products arising from spermine oxidation are not the sole factors responsible for the inhibition of cell growth by spermine, since Higgins et al. (1969) obtained inhibition in KB cells by spermine in the presence of some sera with no spermine oxidase activity. In addition, the cytotoxic effect of polyamines-to be precise, of their oxidatively deaminated products-is not the same for every cell type. It has been proved that oxidized spermidine is cytotoxic in the presence of calf serum only to normal cells, while polyoma virustransformed cells, adenovirus type 1Ztransformed cells, or spontaneously transformed cells are considerably more resistant (Otsuka, 1971). A “spermidine index” for a cell culture, defined as the highest level of spermidine that does not have cytotoxic effects in a standard test system, has been proposed (Otsuka, 1971).This index measures the ability of a cell culture or line to neutralize the cytotoxic effects of spermidine (Otsuka, 1971). The cytotoxic effects of spermidine can be neutralized by the serum’s albumin content, which adsorbs spermidine molecules (Otsuka, 1971). Dioxidized spermine, either in its free cationic form or bound to an unidentified serum component, potently arrested cell proliferation in the GI phase of the cell cycle (Gaugas and Dewey, 1979; Gaugas, 1980). This occurs when polyamines are added to cultures of murine leukemic T lymphocytes, of human leukemic lymphocytes, or of Harding-Passey mouse melanoma and is probably a consequence of the interaction of polyamine oxidase with the exogenous polyamines (Gaugas and Dewey, 1979; Gaugas, 1980). Addition of the diamines putrescine and cadaverine did not produce inhibition (Gaugas and Dewey, 1979). The fact that the cells were arrested in the GI phase suggests that this inhibition was not due to acrolein, which is instantaneously cytotoxic at all phases of the cell cycle (Gaugas and Dewey,

1979).

As has been previously reported (see Section I,E,l, Part I, Vol. 35), the oxidation products of polyamines are aminoaldehydes, which can react with thiol groups. Addition of thiols to culture containing amine oxidase in the medium can protect against polyamine toxicity. This was found in a cell line derived from Harding-Passey mouse melanoma, which was protected against spermidine toxicity by addition of L-cysteine (Dewey and Gaugas, 1980).This protective effect is specific for L-cysteine, because the unnatural isomer D-cysteine enhances the inhibition by spermidine (Dewey and Gaugas, 1980). There is some relationship between bound serum aldehydes and cell growth. In the sera of patients with early malignancies, the amount

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of bound aldehyde that could be released by treating the serum with As203is significantly lower than that in normal sera, but, as metastases occur, it rises above normal values (Quash and Maharaj, 1970). This difference no longer exists when the patients with malignant disease have responded to an antineoplastic treatment (Quash and Maharaj, 1970). Administration of nontoxic doses of putrescine along with the 3,4benzopyrene almost completely prevented tumor development in mice (Kallistratos, 1975a,b; Kallistratos and Fasske, 1976). Although the mechanism of this inhibition of carcinogenesis is still unknown, it is probable that putrescine comes between 3,4-benzopyrene and cell components, hindering binding of the carcinogen (Kallistratos, 197513). Topically applied putrescine inhibits both the induction of epidermal ODC activity and the promotion of mouse skin tumors by TPA (Weekes et al., 1980). This inhibition is not due to some general cytotoxic effect of the diamine, since the application of putrescine did not inhibit the induction of activity of SAMD by TPA (Weekes et al., 1980). Treatment with putrescine before TPA application has little effect (Weekes et al., 1980). It is not clear whether putrescine regulates ODC activity by decreasing its rate of synthesis or by accelerating its rate of degradation. It has been shown that putrescine does not inhibit TPA-induced epidermal ODC activity via production of ODC antizyme (Weekes et al., 1980). This was true also for a clone of rat hepatoma cells in culture (McCann et al., 1979a). Therefore, there might be two separate mechanisms for ODC regulation by putrescine: one through the induction of the inhibitory antizyme that complexes with the enzyme (see Section I,B,l of Part I, Vol. 35 and of Section I,A of this volume) and another without induction of any significant amount of antizyme (McCann et al., 1979a; Weekes et al., 1980).Antizyme induction or noninduction depends on the concentration of putrescine in a rat hepatoma cell line partially resistant to a-methylornithine. Low concentrations of putrescine have an effect on ODC activity similar to that seen of general inhibitor of protein synthesis cycloheximide, i.e., block of new ODC synthesis (McCann et al., 1979a). On the contrary, high concentrations of putrescine elicit the induction of ODC antizyme (McCann et al., 1979a). In spite of the results of McCann, the comparable dose response curves for inhibition of endogenous ODC and for induction of ODC antizyme by diamines and polyamines in HeLa cells suggest that in this culture system there is no need to invoke the existence of separate and distinct mechanisms for regulation of ODC activity (Branca and

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Herbst, 1980).The induction of ODC activity elicited in HeLa cells by arginine was diminished by increasing amounts of canavanine, an arginine analog, in the medium (Prouty, 1976a). Ornithine has an effect analogous to that of putrescine in decreasing tumor incidence. Addition of this amino acid to water and food decreases tumor incidence in mice inoculated with either transplantable breast adenocarcinoma (C3HBA) or MuSV-M virus (Rettura et al., 1978). The mechanism by which ornithine exerts its antitumor action is also not known. It was found that the naturally occurring polyamines potentiate thermal inactivation of mammalian cells (see Section 1,A). The combined effect of heat and polyamines on transplanted B16 melanomas was studied in mice (Hazan, 1980). Synergistic interactions between heat and spermine or cadaverine were demonstrated, but the effects were weak because of the toxicity of the polyamines, which prevent giving high doses (Hazan, 1980).These data agree with those of studies on cultured Chinese hamster cells (Ben-Hur et al., 1978)(see Section I,A), and there is a good correspondence between the in vitro and in vivo results. An interesting effect of spermine was reported by Beck (1977), who found that addition of the tetraamine to cultures of human Wilms’s tumor or rat hepatoma cells reduces the cytotoxic effects of vincristine and vinblastine, which interact with microtubule structures. Vinblastine, vincristine, and colchicine, all of which possess microtubuledisrupting activity, also inhibit ODC induction by TPA in mouse skin (O’Brien et al., 1976). Last, among the different ways by which one or more polyamines might exert their inhibiting effects on cell growth, we must consider the compounds formed by polyamines with other chemical substances. Some of these compounds, the acridines connected by the naturally occurring polyamines to form putrescine diacridine, spermidine diacridine, and spermine diacridine, were studied, and their effects on the growth of HeLa cells and of P388 and L1210 leukemia cells were compared to those of the parent compound 9-aminoacridine (Canellakis and Bellantone, 1976). The diacridines are more effective growth inhibitors than 9-aminoacridine, and the inhibition is not due to the toxicity of oxidized polyamines formed in the medium, since it is seen with leukemic cells P388 and L1210 grown in the presence of horse serum, which does not contain polyamine oxidase (see above). In the case of HeLa cells, which are grown in a medium containing calf serum, the number of cells in the presence of spermine diacridine reaches a plateau and remains at this plateau for many days, while

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exposure of cells to 9-aminoacridine for the same periods of time and under identical conditions results in slower rates of growth but not in a complete arrest of the growth of the cells (Canellakis and Bellantone, 1976). Synthesis of RNA is inhibited immediately, and synthesis of protein and DNA is inhibited later in exposure to both 9-aminoacridine and spermine diacridine (Canellakis and Bellantone, 1976). When P-388 and L1210 cells were exposed to spermine diacridine and then inoculated intraperitoneally (i.p.) into mice, cells that would have been inhibited from subsequent growth in culture can grow when placed in the animal (Canellakis and Bellantone, 1976). In culture, however, spermine or spermidine can reverse the inhibition, probably by the displacement of endogenous bound diacridines (Canellakis and Bellantone, 1976). Another series of compounds, the ferrocenyl polyamines, was screened for antitumor activity against lymphocytic leukemia P388 (Fiorina et al., 1978). These compounds were synthesized with the intent to produce materials that would interact strongly with the tumor surface nucleic acids and, through the hapten portion of the molecule, stimulate antibody formation. The target ferrocenyl polyamines were inactive, but the R-substituted compounds, such as diamide, triamide, and tetraamide, exhibited low, but significant, antitumor activity (Fiorina et al., 1978). It appears that the incorporation of the ferrocene group into an appropriate polyamide carrier might provide an agent with enhanced antitumor activity (Fiorina et al., 1978). Israel et al. (1964) synthesized a series of substances related to spermine and spermidine that showed significant antitumor activity in viuo against four transplantable mouse tuniors-L1210 ascitic lymphatic leukemia, P1534 lymphatic leukemia, C1498 myelogenous leukemia, and DBRB mammary carcinoma. Against human epidermoid carcinoma KB cells in a culture system containing calf serum, the triamines and tetraaniines synthesized demonstrated, in general, the same high degree of inhibitory activity as spermine and sperniidine (Israel et al., 1964). Furthermore, in a systematic examination for growth-inhibitory activity against KB cells, it was found that incorporation of a 2-aminoethyl terminal group in polyamine derivatives is essential for inhibitory activity (Israel and Modest, 1971). Another product, a homolog of spermine, N,N’-bis(3-aminopropyl)nonane-1,9dianiine, when administered in the form of its tetrahydrochloride, inhibits the growth of a variety of leukemias and solid tumors in mice, rats, and hamsters (Israel et ul., 1973). This compound also requires bovine plasma amine oxidase and oxidative deamination, as shown by its not being inhibitory against KB cells in horse serum (Israel et

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al., 1973). It should be noted that the dialdehyde products of this compound inhibited the growth of KB cells in vitro both in the presence and in the absence of bovine plasma amine oxidase and without a need for generation of acrolein (Israel et al., 1973). The effect of some steroidal amines (irehdiamine A, malonetine, 3a-amino-5a-androstane, and 3a-amino-5a-pregnane) on membrane structure and permeability has been investigated in human KB cells (Silver et al., 1970). Some of these amines caused a rapid loss from the cells of more than 95% of accumulated **Mg,but this effect is not a characteristic of tumor cells, since normal mouse fibroblasts are also sensitive to these substances to approximately the same extent (Silver et al., 1970). This membrane effect, although it has been found to be a property of these polyamines not present in mammals, could indicate that spermidine and spermine inhibited growth of KB cells in culture (Israel et al., 1964; Higgins et al., 1969) in the same way.

B. PHARMACOLOGICAL INHIBITORS 1. Znhibitors of ODC Most experimental efforts to deplete cellular polyamines have been focused on the development of inhibitors of the rate-limiting enzyme ODC. Among these are diamines, such as 1,3-propanediamine, 1,5pentanediamine, and 1,6-hexanediamine. These diamines dramatically decreased both tumor growth and ODC activity in neuroblastoma cells, whereas glioma cells continued to grow (Chapman and Glant, 1980). Differences in sensitivity to growth inhibition probably are not due to differences in drug accessibility since the enzyme was inhibited in both cell lines (Chapman and Glant, 1980). The ODC activity in C1300 neuroblastoma was also inhibited significantly in the presence of cytolytic concentrations of bromoacetylcholine and bromoacetate (Chapman et al., 1978), which inhibited cell replication, and RNA and protein synthesis in parallel. Because similar inhibition of neuroblastoma cell replication was obtained with the ODC inhibitor 1,3-diaminopropane, it seems that the mechanism of the potent cytolytic action of bromoacetylcholine and of bromoacetate may be related to inhibition of this enzyme (Chapman e t al., 1978). Among the most potent diamines able to inhibit the formation of putrescine and spermidine, Janne and his co-workers described an analog of putrescine, the l,Sdiaminopropane, and some of its deriva-

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tives, such as the hydroxylated derivative 1,3-diamino-2-propanol, which all inhibit ODC activity. Once again, Ehrlich ascites carcinoma cells, which contain substantial amounts of polyamines and have typical growth-dependent fluctuations in the activities of their biosynthetic decarboxylases (see Section 11, Part I, Vol. 35), have been a suitable model system for studies devoted to the elucidation of the role of polyamines in tumor cells. It was demonstrated that the polyamine patterns in mice with Ehrlich ascites carcinoma could be modified by injection of diaminopropane or cadaverine (Kallio e t al., 1977). Disappearance of ODC activity was accompanied by depletion of cellular putrescine and markedly reduced concentrations of spermidine (Kallio et d., 1977). Repeated injections of diaminopropane virtually abolished any increase in ODC activity in the Ehrlich ascites cells (Alhonen-Hongisto et al., 1979b). In contrast to ODC, there were insignificant changes in the activity of SAMD in response to the diamine injections (Kallio et al., 1977; Alhonen-Hongisto et al., 197913). In a further investigation of the diamine-induced inhibition of ODC, derivatives of 1,3-diaminopropane were tested in cultured Ehrlich ascites cells. 1,3-Diamino-2-propanol appeared to be as potent or even more potent an inhibitor of ODC than the parent compound, whereas 1,2-diaminopropane and 1,2-diamino-2-methylpropanewere less active (Alhonen-Hongisto et al., 1979a). During the growth phase of ascites cells, diaminopropanol permanently abolished increases of ODC after dilution of stationary cell cultures with fresh medium, whereas the activity of SAMD was unaffected by the inhibitor (Alhonen-Hongisto et al., 1979a). Exposure of Ehrlich ascites cells to 1,3-diamino-2-propanol resulted in decreased polyamine accumulation and marked disturbances in cell metabolism and proliferation, as judged by impaired syntheses of DNA and protein and decreases in cell numbers (Alhonen-Hongisto et al., 1979a, 1980a) or cell mass (Alhonen-Hongisto et al., 1979b). Inhibition of DNA and protein synthesis occurred after depletion of spermidine and spermine has been established (Alhonen-Hongisto et al., 1979a). Severe inhibition of polyamine accumulation and of cell proliferation by diaminopropanol was seen in cultures of HeLa cells, as well induced the (Branca and Herbst, 1980). 1,3-Diamino-2-hydroxyropane formation of ODC antizyme, and, like other diamines that cannot serve as precursors of the naturally occurring polyamines, decreased intracellular concentrations of the polyamines, which in turn blocked the proliferation of the HeLa cells. The antiproliferative effect of the ODC

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antizyme inducer does not destroy the viability of the arrested culture, since cell replication occurs after removal of the antizyme inducer from the media (Branca and Herbst, 1980). Although it is strongly suggestive, close parallelism between druginduced growth inhibition and a decrease in polyamine content cannot be taken as definitive proof that inhibition of polyamine synthesis alone is responsible for the antiproliferative effects of the drug. The most straightforward evidence that an inhibitor is specific for the prevention of polyamine synthesis is the reversal of the antiproliferative action of the drug b y exogenous polyamines. Reversal experiments are, however, complicated by the fact that inhibitors of polyamine synthesis may interfere with the cellular uptake of natural polyamines. In fact, tumor cells depleted of putrescine and spermidine take up extracellular polyamines much more effectively than do untreated cells (Alhonen-Hongisto et al., 1980b). In contrast with the data demonstrating a causal relationship between depletion of polyamines and inhibition of DNA synthesis (Alhonen-Hongisto et al., 1979a), it has been found that despite its more pronounced inhibition of polyamine accumulation diaminopropanol has less striking antiproliferative action than the diguanidines (Alhonen-Hongisto et al., 1980a). In addition, the inhibition of DNA and protein synthesis by diaminopropanol was diminished but not abolished by simultaneous addition of putrescine to the culture medium (Alhonen-Hongisto et al., 1979a). The inability to completely reverse the action of diaminopropanol on cell growth with natural polyamines was apparently due to the fact that it is remarkably difficult or even impossible to increase intracellular polyamine concentrations by adding exogenous polyamines in the presence of the inhibitor. Nevertheless, the diaminopropanol-induced arrest of growth is reversible, as judged from the rapid increase in ODC activity followed by restoration of DNA synthesis (Alhonen-Hongisto et al., 1979a). It has been found that 1,3-diaminopropane also induces ODC antizyme in rat hepatoma cells and, like putrescine, can act on ODC by two distinct regulatory mechanisms (McCann et al., 1977a, 1980). Various congeners of ornithine have been found to be potent inhibitors of ODC activity in eukaryotic cells. The first inhibitor of polyamine synthesis tested for elucidation of the metabolic consequences of inhibition of putrescine synthesis, was a-hydrazinoornithine. This drug prevents the increase in intracellular putrescine that occurs when hepatoma cells grown to high density in culture are diluted with fresh medium (Harik et al., 1974a). Doses of the drug that markedly reduce putrescine do not appreciably affect the syn-

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thesis of either RNA or DNA (Harik et al., 1974a). Surprisingly enough, it was also shown that when added to the rat hepatoma cells at the time of dilution with medium, a-hydrazino-ornithine evoked a dose-related increase in ODC activity as much as threefold and prolonged the apparent half-life of the enzyme from 10 min to 28 min (Harik et al., 1974b). The racemic form of a-hydrazino-ornithine, DL-a-hydrazino-6aminovaleric acid (DL-HAVA),appeared to be a potent and fairly specific inhibitor of ODC in transplanted sarcoma 180 in mice (Kato et ul., 1976). This drug greatly suppressed putrescine concentration and its formation from ornithine but did not significantly affect the concentrations of spermidine and spermine (Kato et ul., 1976). On the contrary, DL-HAVA efficiently prevents the accumulation of spermidine and spermine in BKT-1 tumor cells (Raina et al., 1978). A single i.p. injection of DL-HAVA into mice bearing sarcoma 180 also caused strong inhibition of DNA synthesis, which was reversed by administration of putrescine, but not of cadaverine or 1,7-diaminoheptane (Kato et al., 1976). Abdel-Monem and his co-workers (1975a) found that ODC was strongly inhibited in L1210 leukemic cells by a-methylornithine, another competitive inhibitor of the enzyme. Complete disappearance of putrescine and a 50% decrease in spermidine content not accompanied by inhibition of growth were evident in these cells treated with a-methylornithine for two generations (Newton and Abdel-Monem, 1977), whereas in zjiuo administration of the drug to mice with L1210 leukemic cells did not alter the increases in polyamine levels normally observed during tumor growth (Weeks and Abdel-Monem, 1977). There was also no in uiuo effect of the tert-butyl ester of a-methylornithine (Weeks and Abdel-Monem, 1977). In uitro, a-methylornithine did not alter DNA synthesis, indicating that a large portion of the polyamines are not essential for cellular growth in these cells (Newton and Abdel-Monem, 1977). However, a later study of Mamont et al. (1978a) showed that a-methylornithine does inhibit L1210 cell proliferation. a-Methylornithine markedly inhibited the proliferation also of Bri8 human leukemic lymphocytes, not the parent cells present at the onset of incubation but only the daughter cells and their progeny (Gaugas, 1980).The inhibitory effect could be prevented b y adding putrescine or other biogenic polyamines (Gaugas, 1980). DL-a-Methylornithine prevented the biphasic increases in putrescine concentration in cultures of rat hepatoma cells induced to proliferate by dilution with fresh medium (Mamont et al., 1976, 1978c) and blocked the proliferation of rat hepatoma cells and mouse leukemia

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L1210 cells in culture (Mamont et d., 1978a). Increases in cellular spermidine concentration were prevented as well, and inhibition of DNA synthesis and cell division was observed (Mamont et al., 1976). Addition of polyamines results in an immediate resumption of cell proliferation, which is also restored by L-ornithine, presumably due to in situ competitive inhibition of ODC (Mamont et al., 1976, 1 9 7 8 ~ ) . Also in this case, the specificity of the reversing effects of the polyamines was confirmed by the finding that neither cadaverine nor l,Sdiaminopropane, the higher and the lower homologs of putrescine, could overcome the inhibition of cell proliferation by a-methylornithine (Mamont et al., 1976). Another effect of a-methylornithine which was reversed by spermidine is inhibition of cytokinesis with induction of the formation of binucleate HeLa cells (Sunkara et al., 1979a). Treatment of 9L rat brain tumor cells with a-methylornithine resulted in cytostasis when the cells were plated in monolayer cultures at an initial cell density of 5 x 105 per flask but not of 1 x 10' (Seidenfeld and Marton, 1980). This could be explained by the fact that the amount of a-methylornithine entering the cells depends only on its concentration in the medium. As in hepatoma cells and in mouse leukemic cells, the inhibition of 9L cell proliferation by the drug appears to be a specific result of polyamine depletion and can reversed by addition of exogenous putrescine to the culture medium (Seidenfeld and Marton, 1980). a-Methylornithine did not prevent the cells from initiating DNA synthesis. Thus, in these cells too increases in ODC activity and in intracellular polyamine content do not appear to be the signals for initiating DNA synthesis (Seidenfeld and Marton, 1980). Despite the fact that a-methylornithine has been found markedly to inhibit growth of rat hepatoma cells and mouse L1210 cells, it did not affect the growth pattern of Ehrlich ascites tumor cells grown in culture (Oredsson et al., 1980a). A possible explanation is that the latter have putrescine and spermidine contents that are considerably higher than those of the other neoplastic cells. On the other hand, a-methylornithine induced a significant increase in vivo in the cell lethality of Ehrlich ascites tumor cells, suggesting that the drug produces its antiproliferative effect, at least in part, by cytocidal action (Linden et al., 1980). When added to hepatoma cells in culture, a-methylornithine causes an increase in ODC activity, probably stabilizing the enzyme and slowing its degradation (McCann et al., 1977b). This is a common disadvantage of all competitive inhibitors of ODC, which stabilize the

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enzyme in both cultured cells and in uiuo (Harik et al., 1974b), probably because the enzyme bound to the inhibitor is less susceptible to degradation than the free enzyme. This binding to the enzyme and this protection from degradation, resulting in an increase in the half-life of ODC, was also found in neuroblastoma and glioma cells in culture after addition of a-methylornithine (Chapman and Glant, 1980). A clone of rat hepatoma tissue culture cells with a rate of cell proliferation partially resistant to a-methylornithine has been isolated and designated HMO, (Mamont e t al., 1978b). It appears that the reason for the partial resistance of HMOAcells to the ODC inhibitor is their overproduction of putrescine and spermidine (Mamont et al., 1978b), which lends an initial resistance of these cells to the antiproliferative drugs. Significantly, when spermidine depletion has finally occurred, i.e., within 2.5 to 3 generations, cell growth slows (Mamont et al., 1978b). A series of synthetic structural analogs of ornithine were tested as competitive inhibitors of ODC obtained from rat hepatoma cells in culture (Bey et al., 1978). This study clarified the structural features of L-ornithine that are required for binding to mammalian ODC. There is a requirement for an L configuration of the ligand. (+)-aMethylornithine, which was assigned the L configuration on the basis of rotational criteria, was found to be the most effective inhibitor of ODC (Bey et al., 1978). A primary terminal amino group in the ligand appears to be important for enzyme inhibition, and the distance between the two nitrogen atoms of the ornithine analogs is of major importance for inhibition (Bey et al., 1978). Last, hydrophobic interactions with the side chain of the ligand are important for the binding, since hydrophilic functions on the a- and the P-carbon atoms abolish all affinity for the enzyme (Bey et al., 1978). a-Difluoromethylornithine (DFMO), a dihalogenated derivative of a-methylornithine, is one of the most specific inhibitors of ODC because of its mode of action as an enzyme-activated irreversible inactivator (Mamont et al., 1980; Oredsson et al., 1980a). At variance with a-methylornithine, DFMO proved to be an affective inhibitor of growth of Ehrlich ascites tumor cells in culture (Oredsson et al., 1980a) or in uiuo (Alhonen-Hongisto et al., 1979b). However, even though DFMO is an irreversible inhibitor of ODC, there was not total inhibition of the enzyme activity (Oredsson et al., 1980b), and when the drug was added after the initial surge of ODC activity, growth proceeded as in untreated control cultures (Oredsson et al., 1980a). Once the polyamine content has been significantly lowered by the drug, however, the cells grow slowly and synthesize their mac-

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romolecules very poorly, suggesting that at least putrescine and spermidine are essential for maximum rates of cell proliferation (Oredsson et al., 1980b). Another illustration of a greater effectiveness of DFMO than of a-methylornithine was the finding that DFMO, but not a-methylornithine, inhibited the growth of human prostatic adenoma cells (Mamont et al., 1978a). DFMO decreases the concentrations of putrescine and spermidine, but not of spermine, in rat hepatoma cells (Mamont et al., 1978a,c) and in mouse leukemia cells cultured in vitro (Mamont et al., 1978a) and in Ehrlich ascites cells in vivo (Alhonen-Hongisto et al., 1979b). Cell growth inhibition was partially overcome by L-ornithine (Mamont et al., 1978a,c) or prevented b y polyamines, 1,3-diaminopropane, and cadaverine (Alhonen-Hongisto et al., 1979b). It is worth noting, however, that prolonged treatment of hepatoma cells and L1210 cells with these ODC inhibitors did not completely arrest cell growth, suggesting that depletion of putrescine and spermidine does not totally block the cell cycle (Mamont et al., 1978a,c), although in hepatoma cells the most immediate and the predominant consequence of putrescine and spermidine depletion is a decrease in DNA synthesis, with later depression of RNA and protein synthesis (Mamont et ul., 1978c, 1980). Because an inhibition of L1210 cell growth in culture by DFMO has been demonstrated, the effects of the drug were examined in vivo in mice bearing L1210 leukemia (Prakash et al., 1978). Unlike the reversible inhibitor a-methylornithine, DFMO can prolong the survival of mice (Prakash et al., 1978; Seiler et al., 1978). Like a-methylornithine, DFMO also inhibits cell proliferation of 9L rat brain tumor cells at high concentrations but not at low concentrations (Seidenfeld and Marton, 1979a). Both the high and the low concentrations cause equal degrees of depletion of intracellular putrescine and spermidine content, but have no effect on spermine content (Seidenfeld and Marton, 1979a). This is the first demonstration of continued proliferation of a wild-type cell line at the same rate as controls in spite of depletion of more than 95% of its normal complements of two of the three polyamines. In addition, the lack of correlation of the degree of polyamine depletion with the degree of cytostatic action at the concentrations of DFMO tested implies that inhibition of proliferation by this drug is not due to effects on polyamine content of 9L cells, but rather to some non-ODC specific action (Seidenfeld and Marton, 1979a). No effects on DNA synthesis were noticed when DFMO was given in vivo to L1210 leukemic mice (Seiler et al., 1978) or to mice injected with Ehrlich ascites cells (Alhonen-Hongisto et al., 1979b) or

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in vitro in cells infected with human cytomegalovirus (HCMV) (Isom and Pegg, 1979). Isom and Pegg (1979) found that although HCMVinduced stimulation of ODC was prevented by either a-methylornithine or DFMO, the replication of HCMV was not significantly altered for 8 days. In HeLa cells, DFMO caused a rapid inhibition of growth and arrested a majority of the cells in the S phase (Sunkara et al., 1980). In this case too, the growth inhibition was readily reversible b y an exogenous supply of putrescine (Sunkara et aZ., 1980).An analogous inhibitory effect by DFMO was found in Ehrlich ascites tumor cells, which also were arrested in the S-G, phase of the cycle (Heby et al., 1978b). The growth rate of a murine mammary sarcoma EMTG in tissue culture was slowed by DFMO (Prakash et al., 1980).When mice were inoculated with EMTG cells, administration of DFMO beginning 5 days after tumor inoculation resulted in an 80% inhibition of tumor weight (Prakash et al., 1980). Only minimal cytostatic effects of DFMO were observed in neuroblastoma cultures (Chapman, 1980). The effect of continuous oral administration of DFMO on the growth rate of experimental rat hepatoma 5123 was investigated (Kellen et aZ., 1980).The drug caused a significant retardation of growth rate, but did not completely prevent growth (Kellen et ul., 1980). A phenomenon observed during DFMO-induced spermidine deprivation was the enhancement of SAMD activity (Mamont et al., 1978a,c; Seiler et al., 1978).This explains the continuing accumulation of spermine in cells treated with the drug (Mamont e t al., 1978a,c; Seileret al., 1978; Alhonen-Hongisto et u l . , 1979a,b, 1980a).The enhancement of SAMD by inhibitors of ODC could not be a result of direct stabilization b y the drugs, since the drugs are said not to have any affinity to the enzyme. However, DFMO and dianiinopropane appeared to protect SAMD from normal degradation, seen as a marked prolongation of the half-life of the enzyme (Alhonen-Hongisto, 1980; Alhonen-Hongisto et al., 1980a). As revealed in the kinetic data, the mechanism of action of the drugs also involved an enhanced synthesis of SAMD (Alhonen-Hongisto, 1980). In addition to the enhancement of SAMD activity, another mechanism contributing to the compensation for polyamine deprivation has been observed in Ehrlich ascites cells grown in the presence of DFMO. The disappearance of putrescine and spermidine in the cells was accompanied by an appearance of cadaverine, which was rapidly converted to aininopropylcadaverine, an analog of spermidine (Alhonen-Hongisto and Janne, 1980). Supplementation of DFMO-treated cultures with spermidine abolished the enhanced SAMD activity and prevented the formation of

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cadaverine and its aminopropyl derivative (Alhonen-Hongisto and Janne, 1980), suggesting that the two events may be coordinately regulated by polyamines. Under circumstances in which the accumulation of natural polyamines is inhibited, compounds not normally found in cells are synthesized and accumulated to take over the functions of natural polyamines and to compensate for the polyamine deficiency. a-Acetylenic putrescine, another synthetic irreversible inhibitor of ODC, has been found to be more potent than DFMO in rat hepatoma cells in vitro (Mamont et al., 1980). Other analogs of ornithine than a-methyl-substituted ones have also been found by Abdel-Monem and co-workers (1975a). A number of a-alkyl- and benzylornithine analogs were tested as possible inhibitors of ODC in L1210 leukemic cells. The idea was that since the substitution of the a-hydrogen in the ornithine molecule with a methyl group provided a potent competitive inhibitor of ODC, replacement of the a-hydrogen with other alkyl or aralkyl groups might also provide potent inhibitors of the enzyme. However, these new compounds were very poor inhibitors of the enzyme (Abdel-Monem et al., 1975a). It has been found that tumors of neural origin are sensitive to retinoids. Retinol, retinal, and retinoic acid arrested the proliferation of C1300 neuroblastoma cells in culture (Chapman, 1980; Chapman et al., 1980). Glioma cells were less sensitive to all three analogs (Chapman, 1980; Chapman et al., 1980).A correlation was shown between the ability of retinol to inhibit ODC activity and its potency as an inhibitor of cell proliferation. When vitamin A was tested in combination with DFMO, the antiproliferative effects of the two drugs in both neuroblastoma and glioma cells were additive (Chapman, 1980). In two-stage carcinogenesis, TPA induces a very rapid increase in ODC (see Section III,C, Part I, Vol. 35), with the subsequent accumulation of polyamines. It is therefore of great interest that retinoids have been shown to inhibit these effects of TPA. Verma, Boutwell, and their colleagues found that treatment of mouse skin with retinoic acid, prior or soon after application of the tumor promoter, resulted in a much lesser induction of ODC (Verma and Boutwell, 1977, 1980; Verma et al., 1979). Since the retinoid affected neither enzyme activity in cellfree extracts nor the in vivo production of ODC antizyme, it was suggested that the retinoid interferes with enzyme induction (Verma and Boutwell, 1977).Subsequent studies with other retinoids revealed that 13-cis-retinoic acid, 5,6-dihydroretinoic acid, and two cyclopentenyl analogs of retinoic acid were also potent inhibitors of the development of skin papillomas and that this inhibitory property correlates well with their relative potencies in inhibiting ODC induction (Boutwell

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and Verma, 1978; Verma et al., 1979). 5,6-Epoxyretinoic acid, a biologically active metabolite of retinoic acid, is also active in mouse skin, where it inhibits both the induction of ODC activity and skin tumor Retinoic acid inhibits ODC promotion b y TPA (Verma et al., 1980~). activity induced in normal rat kidney cells not only b y TPA but also by epidermal growth factor and sarcoma growth factor (Paranjpe et al., 1980). When skin tumors were induced by DMBA, retinoic acid failed to inhibit either the induction of ODC activity or the tumor formation (Verma et d.,1980b). This indicates that retinoic acid’s protection against skin carcinogenesis is not universal, since it inhibits skin tumor formation by some agents but not by others. In addition, although retinoic acid is not a skin tumor promoter, when given in conjunction with DMBA treatment it even potentiated the formation of skin papillomas by DMBA (Vermaet al., 1980b). Another proof of the nonuniversality of the protective effect of retinoic acid is the fact that retinoic acid only partially inhibited the induction of ODC by germicidal UV light (Lichti et al., 1979). Formation of skin papillomas after DMBA was inhibited by 7,8benzoflavone, which also effectively inhibited the DMBA-induced ODC activity, but not TPA-induced ODC activity (Verma et al., 1980b). The induction of ODC activity in mouse epidermal cells after TPA treatment has been supposed to have the characteristic of a cell surface receptor-mediated process (see Section III,C, Part I, Vol. 35). Local anesthetics can modify a variety of cellular responses mediated by membrane receptors. On these grounds, local anesthetics such as lidocaine, tetracaine, and procaine, which are tertiary amines specifically acting on polyamine biosynthesis, have been used in order to clarify the role of the polyamine pathway in tumor promotion (Yuspa et al., 1980a,b). When added to mouse epidermal cells in culture, the anesthetics inhibited ODC inducation by TPA. In uiuo, lidocaine essentially abolishes ODC induction only when applied shortly after TPA (Yuspa et al., 1980a,b). These results are consistent with local anesthetics inhibiting at the site of interaction of TPA and its putative epidermal receptor. Local anesthetics also inhibit ODC induction b y UV light, which is probably not membrane mediated (Yuspa et al., 1980a,b). In addition, sulfur mustard, a potent inhibitor of two-stage skin tumorigenesis, did not alter TPA-induced ODC activity in mouse epidermis (De Young et al., 1977). The antipsoriasis drug anthralin has been tested for inhibition of TPA-induced ODC activity. Anthralin alone is an inducer of ODC and a moderate tumor promoter. However, when applied 2 hr before TPA,

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anthralin inhibited ODC induction and the TPA-induced epidermal cell proliferation (De Young et al., 1980).In addition, in a tumor promotion experiment with DMBA as initiator, anthralin given before TPA inhibited the number of tumors per animal. This inhibitory response may be explained by synergistic toxicity or by competition for binding sites b y the weaker tumor promoter anthralin with the stronger promoter TPA, or both (De Young et al., 1980). In contrast to their effects in vivo, anthralin and 7,12-dimethylbenz[a]anthracene have little effect on ODC activity in cultures of newborn mouse epidermal cells (Lichti et al., 1978). Not only anthralin, but also ethylphenylpropiolate inhibited TPA-stimulated ODC by 70% or more, whereas other inflammatory agents such as iodoacetic acid and cantharidin were able to shift the peak time of ODC activity after TPA (Di Giovanni and Hoel, 1980). All four compounds effectively inhibit TPA-promotion of DMBA-initiated skin papillomas (Di Giovanni and Hoel, 1980). A nonpharmacological molecule inhibiting TPA-induced ODC activity is interferon (Sreevalsan et al., 1980). This molecule was able to inhibit the increase in ODC activity in 3T3 cells stimulated b y other agents, such as epidermal growth factor, vasopressin, insulin, or fibroblast-derived growth factor, used either singly or in combination (Sreevalsan et al., 1980). An inhibition of TPA-induced stimulation of DNA-synthesis by interferon was also shown (Sreevalsan et al., 1980). The role of prostaglandins in the induction of ODC activity by TPA has been reviewed (Verma and Boutwell, 1980). A number of inhibitors of prostaglandin synthesis, such as indomethacin, naproxen, flufenamic acid, and acetylsalicylic acid, inhibited ODC induction by TPA in mouse skin (Verma et al., 1977, 1980a). The inhibition was overcome by application of prostaglandins concurrently with the TPA, thus indicating that PGE1, PGE2, PGDZ, and PGIz play roles in the induction of enzyme by TPA. Although prostaglandins mediate ODC induction by TPA in mouse epidermis, application of prostaglandins alone did not induce epidermal ODC activity, suggesting that prostaglandins are necessary but not sufficient for ODC induction by TPA (Verma et al., 1980a). In contrast to many data that have indicated that ODC induction is an essential component of the mechanism of skin tumor promotion (O’Brien et al., 1975a,b; O’Brien, 1976; Verma et al., 1977; Verma and Boutwell, 1980), a recent study suggested that polyamines are possible mediators of tumor promotion and therefore the decreases in polyamine levels better reflect the potencies of inhibitors of tumor promotion (Weeks et al.,1979). Changes in ODC activity after coincident treatment with TPA and either a-methylornithine or fluocinolone acetonide (FA), are not representative of the alterations in the in vivo

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polyamine level changes nor related to effectiveness of tumor promotion inhibition in mouse skin (Weeks et al., 1979). In fact, both a-methylornithine and FA provide paradoxical enhancements of the TPA-induced ODC activity. However, a-methylornithine causes the TPA-stimulated increased epidermal putrescine levels to be lower, and FA specifically inhibits the accumulation of spermidine (Weeks et al., 1979). Among the anti-inflammatory steroids, dexamethasone was shown to have little, if any, inhibiting effect in vivo on TPA-induced ODC activity (Vermaet al., 1977), while FA, the most potent steroidal inhibitor of mouse skin tumor promotion, paradoxically increased the TPAenhancement of ODC activity in vivo and in mouse epidermal cells in culture (Lichti et al., 1977a,b; Yuspaet al., 1978).This enhancement of the TPA-induced ODC activity by FA has been confirmed by other authors (Weeks and Slaga, 1979). On the contrary, in the Sencar mouse, a mouse stock selected for increased sensitivity to carcinogenesis, FA inhibited the TPA-induction of ODC activity (Weeks and Slaga, 1979). In numerous systems, inhibition of cell proliferation follows treatment of cells with polyamine antimetabolites, which seems to indicate that natural polyamines play an essential role in cellular proliferation processes (Janne et al., 1978). Nevertheless, the data available on this field are quite controversial. Administration of DAP and DFMO to mice with Ehrlich ascites carcinoma resulted in markedly less accumulation of putrescine and spermidine, associated with a striking depression of DNA synthesis (Alhonen-Hongisto et al., 1979b). DFMO reduced the concentration of spermidine much more than did DAP, but caused only marginal depression of DNA synthesis (AlhonenHongisto et al., 1979b). This could be explained in terms of increased concentrations of spermine compensating for the loss of spermidine in cells treated with DFMO (Mamont et al., 1978a,c; Alhonen-Hongisto et ul., 1979b). Injection of mice bearing Ehrlich ascites carcinoma 3-aminoguanidine) with 1,l’-[ (methylethanediylidene)dinitrilo]bis-( (MBAG) potentiates the effects of DAP and diaminopropanol in depressing incorporation of thymidine into DNA (Alhonen-Hongisto et d.,1979b). The potentiation of the antiproliferative effects of DAP or diaminopropanol by MBAG seemed to be specific for the diamines, since no such synergism was found when DFMO and MBAG were injected together (Alhonen-Hongisto et al., 1979b). Pertinent to this was the finding that MBAG even reversed instead of potentiating the antiproliferative effects of DFMO in mice bearing EMT6 tumor (Prakash et al., 1980). Comparing the inhibition of polyamine synthesis and of cell prolif-

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eration by diaminopropanol or by some diguanidine derivatives in cultured Ehrlich ascites cells, it gives rise to the ideas that the antiproliferative action of the diguanidines is not entirely based on the polyamine-depleting properties of the drugs and that diaminopropanol partly takes over the functions of natural polyamines in ascites cells (Alhonen-Hongisto et al., 1980a). On the other hand, it has been observed that inhibition of DNA and protein synthesis in Ehrlich ascites cells by diaminopropanol did not become evident before severe polyamine depletion had developed (Alhonen-Hongisto et al., 1979a, 1980a). In L1210 cells treated with a-methylornithine for two generations, DNA content was not altered to any appreciable extent (Newton and Abdel-Monem, 1977), suggesting that putrescine and spermidine are not essential for DNA synthesis in these cells. Inhibition of hepatoma cell polyamine accumulation by a-methylornithine affects neither DNA synthesis nor the mitotic activity during the first round of cell division, but once spermidine depletion has been achieved, there is a striking decrease in DNA synthesis and the cell multiplication rate (Mamont et al., 1976). A complete dissociation between the inhibition of DNA synthesis and ODC activity has been found for the antipromoter steroid fluocinolone acetonide (FA) (Lichti et al., 1977a,b; Yuspa et al., 1978). Under conditions in which FA induces a maximum inhibition of TPAstimulated DNA synthesis in mouse epidermal cell culture, it potentiates promoter-stimulated ODC activity, which is also enhanced when FA is present only during and after the TPA treatment, although FA itself does not significantly stimulate ODC activity (Lichti et al., 1977a,b; Yuspa et al., 1978). Similar results were obtained in uiuo. When FA and TPA are applied simultaneously to mouse skin, a treatment that completely inhibits the tumor promotion process, the steroid prevents promoter-stimulated DNA synthesis, but it paradoxically potentiates the induction of ODC activity observed after promoter treatment (Lichti et al., 1977a,b; Yuspa et al., 1978). In vivo also FA itself did not induce ODC activity (Lichti et al., 1977a,b; Yuspa et al., 1978). The antipromoter steroid FA did not act as an inducer, yet it accelerated and enhanced TPA stimulation of ODC activity (Lichti et al., 1977a,b; Yuspa et al., 1978).This demonstrates that a rise in ODC activity is not enough to trigger the DNA synthesis response unalterably and suggests that FA exerts its antipromoter effect distal to ODC activation, assuming that this enzyme is indeed involved in the tumor promotion process (Lichti et al., 1977a,b; Yuspa et al., 1978).The in vitro FA-mediated prevention of stimulated DNA synthesis after TPA exposure can be reversed by the addition of putrescine to the culture

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medium, suggesting that putrescine and perhaps other polyamines are mediators in tumor promoter-stimulated proliferation and that FA exerts its effect by reducing intracellular polyamine levels (Lichti et al., 1977a,b; Yuspa et al., 1978). 2. Inhibitors of SAMD The changes in polyamine pattern following inhibition of SAMD are often quite different from those obtained after inhibition of ODC. While the latter mainly decreases concentrations of putrescine and spermidine, inhibitors of SAMD deplete cells of spermidine and spermine, usually associated with a paradoxical accumulation of putrescine. The best and the oldest known inhibitor of SAMD, i.e., methylglyoxalbis(guany1hydrazone) (MGBG), was first examined because its antiproliferative activities against human acute myelocytic leukemia and leukemia L1210 were reported by Mihich (1963a,b, 1964, 1965, 1975) and were found to be prevented in vivo by simultaneous treatment with spermidine. A similar antagonistic effect of spermidine against MGBG on L1210 cells was also found in vitro (Pathak and Dave, 1977). The growth of lymphocytic leukemia P388, B82T, and B8174, mast cell leukemia P815, reticular cell sarcoma P329, lymphomas 4, P288, P1798, and sarcoma 180 ascites in mice and of Dunning-Schmidt leukemia in rats was also inhibited by MGBG (Mihich, 1975). However, the drug was not active against some solid sarcomas and carcinomas or against mouse lymphocytic leukemia A&, L4946, and L5178YF(Mihich, 1965).Thus, the majority of the sensitive tumors were Ieukemias. Many aspects of MGBG inhibition of animal tissue SAMD have been described in detail b y Corti et al. (1974), Janne et al. (1978), and Gaugas (1980). The reader is also referred for information on MGBG and its biological effects to several excellent reviews (Mihich, 196313, 1965, 1975; Williams-Ashman et al., 1976). Here we will summarize only the specific actions of MGBG on polyamine biosynthesis in mammalian tumors. The inhibition of SAMD activity by MGBG was competitive with respect to adenosylmethionine. The SAMDs of many normal and malignant tissues were inhibited to about the same extent by MGBG; there was no difference in the inhibition of the enzyme purified from a subline of mouse L1210 leukemia whose growth was sensitive to the drug in vivo, as compared with that of the enzyme purified from another subline whose growth was resistant to MGBG (Corti et d . ,1973, 1974). The drug rapidly increases the putrescine content and de-

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creases the net accumulation of spermidine and spermine content in sensitive (but not in resistant) sublines of L1210 leukemia cells in V ~ V O (Mihich et al., 1974). Since MGBG inhibits equally well SAMD activity from sensitive and resistant L1210 cells, the fact that spermidine levels were not reduced in drug-treated resistant cells would seem to be related to a reduced uptake and retention of drug by these cells (Dave and Caballes, 1973). MGBG has been shown to interfere with the transport of spermidine and spermine (Dave and Caballes, 1973; Dave et al., 1974; Mihich et al., 1974; Seppanen et al., 1980a,b) and with biosynthesis of spermidine (Corti et al., 1974). In addition, polyamines in turn inhibit cellular uptake of the drug (Dave and Caballes, 1973; Mihich et al., 1974; Seppanen e t al., 1980a,b,c), which was remarkably effectively concentrated inside some tumor cells (cultured Ehrlich ascites carcinoma cells, and human lymphocytic leukemia cells both cultured and circulating) (Seppanen et al., 1980a,b,c). In human leukemic cells the uptake of MGBG was critically dependent on their growth rate (Seppanen et d., 1980~). The drug MGBG is a rather specific inhibitor of the SAMD step in polyamine biosynthesis, inasmuch as millimolar concentrations of it do not inhibit mammalian ODC or spermidine and spermine synthases (Corti et al., 1974). However, MGBG markedly stimulates ODC activity in spleens of leukemic mice (Heby et al., 1973). Paradoxically, the spleen SAMD activity was also markedly increased in the same animals, when assayed 24 hr after the injection of MGBG (Heby et al., 1973; Heby and Russell, 1974). In mouse leukemia L1210 cells treated in vivo with MGBG, intracellular pools of spermidine and spermine are considerably depleted (Heby and Russell, 1974; Mihich et al., 1974). In accord with this, the activity of SAMD was profoundly inhibited when crude cytosolic fractions from Ehrlich ascites cells were incubated in the presence of MGBG (Alhonen-Hongisto e t al., 1980a). Because of many obviously nonspecific effects of MGBG in whole animals, it has been used more successfully in neoplastic cell cultures. The drug effectively blocked the accumulation of spermidine and spermine but caused an increased accumulation of putrescine in L1210 cells (Heby et al., 1977; Newton and Abdel-Monem, 1977; Dave et al., 1978). In L1210 cells these changes were accompanied by a greater decrease in the DNA content (Newton and Abdel-Monem, 1977). Since a decrease in the spermidine level similar to that caused by MGBG was found after a-methylornithine treatment, and this drug failed to inhibit DNA synthesis (Newton and Abdel-Monem, 1977), it seems that the inhibition of DNA synthesis by MGBG is not a result of a decrease in the cellular level of spermidine.

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In cultured murine leukemic L1210 cells, MGBG produced selective and extensive ultrastructural damage to mitochondria (MiklesRobertson et al., 1977; Dave et al., 1978). Similar damage has been observed in ascites L1210 cells treated i n vivo with MGBG (Porter et al., 1979),and it is reversible after the cells are removed from the drug. In P288 mouse leukemia and in NALM-1 human chronic myelocytic leukemia cells, MGBG also produced ultrastructural damage nearly identical to that seen in L1210 cells (Mikles-Robertson e t al., 1979). In Bri8 human leukemic lymphocytes, MGBG completely arrests cell proliferation, and once again the arrest could be reversed by the addition of exogenous spermine (Gaugas, 1980).The cells inhibited, as after a-methylornithine, were not the Fo generation but their progeny (Gaugas, 1980). In HeLa cell cultures the decrease in spermidine and spennine levels caused by MGBG preceded a drop in incorporation of labeled thymidine, uridine, and leucine into DNA, RNA, and protein (Krokan and Eriksen, 1977). When putrescine, spennidine, sperinine, or cadaverine was added simultaneously with MGBG, the drug had no detectable effect on the synthesis of macromolecules (Krokan and Eriksen, 1977). The inhibited synthesis of DNA was not restored in nuclei isolated from cells treated with MGBG by addition of spermidine or spermine (Krokan and Eriksen, 1977). MGBG has been found to inhibit cytokinesis and to induce the formation of binucleate cells in a variety of mammalian cells in culture, including HeLa cells, transformed CHO and SV3T3 cells (Sunkara et al., 1978a,b, 1979a).The effects were reversed by spermidine (Sunkara et al., 1978a,b, 1979a). Studies with MGBG have shown that the molecule in the extract of Harding-Passey mouse melanoma that inhibits the proliferation of the same cell line grown in culture is spermidine (Dewey, 1978). In addition, MGBG has been found to be a powerful noncompetitive inhibitor of the enzymic oxidation of sperinidine to acrolein in the same line of cultured melanoma (Dewey, 1979). Treatment of neoplastic cell cultures with MGBG results in different distributions of the cells in the phases of the cell cycle, depending on the cell type. Polyamine-depleted rat brain tumor cells accumulate in the GI phase (Heby et al., 1977, 1978a), while Ehrlich ascites tumor cells accumulate in the S and Gz phases (Heby and Anderson, 1976; Anderson and Heby, 1977; Heby et al., 1978a,b). In contrast to normal rat fibroblasts, which are arrested in the GI phase by MGBG, SV40transformed fibroblasts (Rupniak and Paul, 1978, 1980) and transformed CHO cells, HeLa cells, and SV3T3 cells (Sunkaraet al., 197913)

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continue through the cell cycle, although at slightly slower rates, after inhibition of spermidine and spermine synthesis. These data substantiate the proposal of Rupniak and Paul (1978) that normal cell retains a polyamine-sensitive growth “regulation point” or “restriction point” in GI at which the “polyamine status” of the cell is or is not sufficiently favorable to enable it to enter into and complete a new cell cycle. In contrast, this polyamine-dependent regulatory mechanism is largely lost in tumorigenic cells. The compound 1,l’-[(methylethanediy1idene)dinitrilolbis(3-aminoguanidine) (MBAG), closely related to MGBG, retains the irreversible inhibitory activity of the parent compound on SAMD activity in Ehrlich ascites tumor cells (Alhonen-Hongisto et al., 1979b) and in mouse mammary EMTG cells (Prakash et al., 1980). MBAG, like MGBG, is an extremely potent inhibitor of DAO in Ehrlich ascites tumor cells (Alhonen-Hongisto et al., 197913). According to Corti and co-workers (1974), two other derivatives of MGBG, dimethylglyoxalbis(guany1hydrazone)and di-N” ’-methylglyoxalbis(guany1hydrazone)also strongly inhibited SAMD in vitro from L1210 cells. has been found to The adenosine analog 9-P-D-xylofuranosyladenine be a competitive inhibitor of SAM synthesis in L1210 cells (Glazer and Peale, 1979). 3. Combination of lnhibitors of ODC and SAMD Agents that have been shown to be potent inhibitors of ODC or SAMD have been combined to achieve simultaneous cellular deprivation of all three chief polyamines. In addition, some unwanted effects of the single drug can sometimes be avoided when the drugs are administered together. The addition of MBAG and a-DFMO to the drinking water of mice with mammary EMTG tumors resulted in an unexpected restoration of normal putrescine and spermidine concentrations in the tumor cells (Prakash et aZ., 1980). MBAG also antagonizes the effect of the ODC-inhibitor on tumor growth (Prakash et al., 1980).In combination with DAP, or diaminopropanol, but not with DFMO, MBAG exhibited profound inhibition of DNA synthesis in Ehrlich ascites cells (Alhonen-Hongisto et al., 1979b).

4. lnhibitors of Spermidine and Spermine Synthases The synthesis of specific inhibitors of spermidine and spermine synthases is greatly hampered by the fact that the catalytic mecha-

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nisms of these enzymes are not precisely known. However, some progress in this field has been made. In addition to finding that putrescine acts as a natural inhibitor of spermine synthase (Kallio et al., 1977), some unphysiological diamines have been shown to inhibit spermidine and spermine synthases in the presence of their natural substrates. Cadaverine appeared to compete with putrescine in the synthesis of spermidine, while a lesser effect on spermine synthase was observed in Ehrlich ascites cells (Kallio et al., 1977). 5. Miscellaneous Molecules

A minimal cellular level of glutathione has been found to be required for ODC activity and polyamine synthesis. When diazenedicarboxylic acid bis(N,N-dimethylamide), a relatively specific oxidant of CSH, is added to cultures of H35 rat hepatoma cells, it causes a fall in the cellular level of GSH and inhibits the stimulation of ODC activity b y serum (Beck, 1978). Exogenous CSH can reverse the diamide effect (Beck, 1978). The effect of leupeptin, a microbial protease inhibitor, on carcinogenesis was examined during the early stage of tumorigenesis induced by DMBA and croton oil (Coto e t al., 1980). Leupeptin inhibited tumor development and the increase in spennidine content in treated mice, probably because it protects a protein inhibitor of ODC from destruction by protease, which is increased by croton oil. Tumor necrosis factor (TNF), a substance known to have anticancer activity against murine meth A tumors in uiuo and human melanoma in cell culture, has been found to inhibit ODC activity when injected into mice or added to melanoma cells in culture (Helson et al., 1977). Because T N F did not change the ODC activity in the assay when added to the reaction mixture nor affect ODC activity in the spleens of TNF-treated mice (Helson et al., 1977), the measurement of ODC is a mirror of the changes in tumor proliferative activity and of the specificity of an antitumor activity. Tosylphenylalanine chloromethylketone (Tos-PheCHzCl) and tosyllysine chloromethylketone (Tos-LysCH2C1)stimulate in cultured rat hepatoma the loss of ODC that follows inhibition of protein synthesis (McIlhinney and Hogan, 1974). A single dose of cis-dichloro(dipyridine)platinum can inhibit the elevation in ODC activity that occurs during development of the L1210 leukemia in mice (Morns et al., 1976). Finally, there are reports that the concentrations of polyamines and the activities of their biosynthetic decarboxylases in mouse L1210 leu-

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kemia cells (Russell, 1972; Heby and Russell, 1973a,b) and in a methylcholanthrene-induced fibrosarcoma in mice (Sukumar and Nagarajan, 1978) are lowered by administration of antineoplastic drugs such as methotrexate, cytosine arabinoside, and 5-azacytidine. But no evidence was provided by these studies that such cancer chemotherapeutic agents directly inhibited any of the polyamine biosynthetic enzymes in the tumor, and the alterations in polyamine formation evoked by these drugs seem to be indirect. V. Concluding Remarks and Speculations

The reader who hoped to find some total differences between the polyamine metabolisms of normal and neoplastic tissues and thereby some promising perspectives for study of both human and experimental oncology sees that none have been found as yet. In spite of this, some comments can be made at this time. The investigation of the links between polyamines and tumors can be divided historically into two facets; in the first of these, quantitative differences between the polyamine levels of neoplasms and those of normals were looked for, and in the second, which began more recently, differences between neoplasms and normals in polyamine biosynthesis regulation were investigated. This dichotomy is quite recurrent in oncology, representing almost a general trend in this discipline. We think that research into the qualitative differences of several aspects of polyamine biosynthesis regulation is the most promising at present and in the near future, since the principal characteristic of the phenotype of neoplasms is a lack, or at least an alteration, of the regulation of some metabolic activities and pathways, including those for polyamines. Qualitative differents have been demonstrated in etiologically different types of tumors, both human and experimental. In several experimental tumors or cultured neoplastic cells, isozymes of ODC or SAM synthetases have been identified. A lack of ODC antizyme formation, loss of the ODC circadian rhythm, decreased responsiveness of ODC to putrescine inhibition, and an imbalance in ornithine metabolism have also been found. It is quite hard to believe, although it is b y no means theoretically impossible, that all these qualitative derangements may turn out in the near future to be a general rule for all the neoplasms. However, all these results are phenomenological descriptions of some specific neoplastic abnormalities, and we cannot be satisfied until we know their causes. Nonetheless, the picture obtained when we assemble these single results might well be helpful for indicating the causes of these abnor-

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malities. Therefore, we think that all these qualitative differences merit maximal attention from researchers and that they will have great importance in the near future, when other analogous findings have been added to our knowledge of polyamines and tumors. At present, in spite of the decades of research, we are well aware that most of the role of polyamines in eliciting the peculiar neoplastic behavior and in permitting invasive neoplastic growth remains almost totally obscure. It is tempting to speculate that polyamines are one of the tools, but by no means the only one, available to the neoplastic cells for their uncontrolled growth. It is consistent with this view that the increases in the activities of the two polyamine biosynthetic decarboxylases can very frequently, although not always, be correlated with the degree of malignancy of certain experimental or human tumors, but not at all with the neoplastic status per se. Furthermore, another quantitative aspect to be looked into further in human research is the changes in the amounts of polyamine derivatives, and therefore the changes in their ratios, in physiological fluids from oncopathic subjects, as compared to subjects with other diseases. In addition, it is once again quite hard to accept, although it is by no means theoretically impossible, that studies of polyamines alone will be a conclusive turning point in solving the oncological puzzle. In this regard, it seems to us that studies of the connections between polyamines and pericellular fibronectin would be very interesting and stimulating and might possibly explain some features of neoplastic behavior. Greater integration of the different fields of oncological research are not only desirable but mandatory. Finally, we realize that studies of polyamines and tumors have disclosed a number of problems much larger than the number solved. Therefore, if this review becomes obsolete in a short time, it will mean that new studies have appeared and have introduced new trends and new perspectives about the connections between polyamines and neoplasms. We hope that the result of our present toil will at least evoke new ideas on this topic. ACKNOWLEDGMENTS

First, we are very grateful to Professor S. Weinhouse (Philadelphia) for his understanding of our delays. Thereafter w e wish to thank those authors who kindly sent 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 (Strasburg), Dr. P. McCann (Cincinnati), Dr. D. Morris (Washington), Dr. K. Nishioka (Hous-

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ton), Dr. G. Quash (Lyons),Dr. A. M. Roch (Lyons), Dr. N. Seiler (Strasburg),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 thank also 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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