Báo cáo khóa học: Genetic approaches to the cellular functions of polyamines in mammals potx

REVIEW ARTICLE
Genetic approaches to the cellular functions of polyamines
in mammals
Juhani Ja¨ nne, Leena Alhonen, Marko Pietila¨ and Tuomo A. Keina¨nen
A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
The polyamines putrescine, spermidine and spermine are
organic cations shown to participate in a bewildering num-
ber of cellular reactions, yet their exact functions in inter-
mediary metabolism and specific interactions with cellular
components remain largely elusive. Pharmacological inter-
ventions have demonstrated convincingly that a steady
supply of these compounds is a prerequisite for cell prolif-
eration to occur. The last decade has witnessed the appear-
ance of a substantial number of studies, in which genetic
engineering of polyamine metabolism in transgenic rodents
has been employed to unravel their cellular functions.
Transgenic activation of polyamine biosynthesis through an
overexpression of their biosynthetic enzymes has assigned
specific roles for these compounds in spermatogenesis, skin
physiology, promotion of tumorigenesis and organ hyper-
trophy as well as neuronal protection. Transgenic activa-
tion of polyamine catabolism not only profoundly disturbs
polyamine homeostasis in most tissues, but also creates a
complex phenotype affecting skin, female fertility, fat depots,
pancreatic integrity and regenerative growth. Transgenic
expression of ornithine decarboxylase antizyme has sugges-
ted that this unique protein may act as a general tumor
suppressor. Homozygous deficiency of the key biosynthetic
enzymes of the polyamines, ornithine and S-adenosyl-
methionine decarboxylase, as achieved through targeted
disruption of their genes, is not compatible with murine

embryogenesis. Finally, the first reports of human diseases
apparently caused by mutations or rearrangements of the
genes involved in polyamine metabolism have appeared.
Keywords: antizyme; ornithine decarboxylase; putrescine;
spermidine/spermine N
1
-acetyltransferase; spermidine; sper-
mine; transgenic mouse; transgenic rat.
Introduction
The cellular functions of the natural polyamines (putrescine,
spermidine and spermine) are still largely unknown,
although a vast number of studies have shown that these
polycationic compounds are crucial to the growth and
proliferation of mammalian cells. Pharmacological approa-
ches are applied typically in studies aimed to unravel their
functions in cellular metabolism and, admittedly, much
valuable information has been generated with the use of
specific inhibitors of polyamine biosynthesis. However, the
last decade has produced a substantial number of experi-
mental studies in which genetic engineering of polyamine
metabolism has been used as a tool to elucidate their cellular
functions. Studies with genetically engineered mice and rats
have not only brought entirely new information about the
involvement of polyamines in various physiological and
pathophysiological processes but they have likewise chal-
lenged some of the conventional wisdoms. Mainly, four
different approaches have been applied in the genetic
engineering of experimental animals: (a) activation of
polyamine biosynthesis through the overexpression of their
biosynthetic enzymes; (b) activation of polyamine catabol-

ism through the overexpression of the enzymes involved
in their catabolism; (c) transgenic expression of ornithine
decarboxylase (ODC) antizyme, a protein inhibiting ODC
activity and facilitating its degradation and (d) gene-
disruption technology applied both to the biosynthetic
and catabolic enzymes.
Polyamine metabolism
Figure 1 outlines the metabolism of the polyamines in a
mammalian cell. Two amino acids,
L
-ornithine and
L
-methionine, are the primary precursors of the poly-
amines.
L
-Ornithine is cleaved from
L
-arginine by mito-
chondrial arginase II [1] or derived from the diet and
L
-methionine is first converted to S-adenosyl-
L
-methionine
(AdoMet). Both ornithine and AdoMet are subsequently
decarboxylated by two cytosolic decarboxylases, namely
ornithine decarboxylase (ODC) and AdoMet decarboxy-
lase (AdoMetDC). The former reaction yields putrescine
Correspondence to J. Ja
¨
nne, A.I. Virtanen Institute for Molecular

Sciences, University of Kuopio, PO Box 1627, FIN-70211, Kuopio,
Finland. Fax: + 358 17 163025, Tel.: + 358 17 163049,
E-mail: Juhani.Janne@uku.fi
Abbreviations: ODC, ornithine decarboxylase; AdoMetDC, S-adeno-
sylmethionine decarboxylase; dcAdoMet, decarboxylated adeno-
sylmethionine; SSAT, spermidine/spermine N
1
-acetyltransferase;
PAO, polyamine oxidase; SMO, spermine oxidase; DENSPM,
N
1
,N
11
-diethylnorspermine; DFMO, 2-difluoromethylornithine;
MGBG, methylglyoxal bis(guanylhydrazone); NMDA, N-methyl-
D
-aspartate; GABA, c-aminobutyric acid; eIF5A, eukaryotic
initiation factor 5 A.
(Received 12 December 2003, revised 19 January 2004,
accepted 22 January 2004)
Eur. J. Biochem. 271, 877–894 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04009.x
(NH
2
CH
2
CH
2
CH
2
CH

2
NH
2
) and the latter reaction
decarboxylated AdoMet (dcAdoMet). DcAdoMet do-
nates its aminopropyl group either to putrescine in a
reaction catalyzed by a transferase, spermidine synthase
to yield spermidine (NH
2
CH
2
CH
2
CH
2
NHCH
2
CH
2
CH
2
CH
2
NH
2
), or to spermidine catalyzed by a separate
transferase, spermine synthase to yield spermine
(NH
2
CH

2
CH
2
CH
2
NHCH
2
CH
2
CH
2
CH
2
NHCH
2
CH
2
CH
2
NH
2
). As the decarboxylation and propylamine
transferase reactions are practically irreversible, an
entirely distinct system exists to convert the higher
polyamines back to putrescine. This system utilizes two
different enzymes, a cytosolic spermidine/spermine
N
1
-acetyltransferase (SSAT) [2] and a peroxisomal flav-
oprotein polyamine oxidase (PAO) [3]. As PAO strongly

prefers acetylated polyamines as the substrates [3,4],
SSAT is the rate-controlling enzyme in this backconver-
sion pathway [4]. As indicated in Fig. 1, spermine can be
either monoacetylated or diacetylated [5] by SSAT. As
seen, diacetylation of spermine would not require a re-
entry of spermidine back to the peroxisome but putres-
cine would be formed via N
1
-acetylspermidine from
diacetylated spermine (Fig. 1). In addition to putrescine
and spermidine, the PAO reaction also yields hydrogen
peroxide and acetaminopropanal. Working with SSAT-
deficient mouse embryonic stem cells, we found that
SSAT is absolutely necessary for the conversion of
spermidine to putrescine while spermine is readily
converted to spermidine in the absence of SSAT [6].
The conversion of spermine back to spermidine in the
absence of SSAT activity is obviously attributable to a
recently discovered amine oxidase, which, when first
cloned was thought to be PAO [7], but was soon
identified as a novel spermine oxidase (SMO) [8]. Unlike
PAO, SMO strongly prefers spermine as the substrate
over its acetylated derivatives and spermidine is not a
substrate at all [8,9]. Figure 1 likewise highlights an
important Ôside trackÕ of spermidine metabolism, namely
it serves as a precursor for hypusine synthesis. This
unusual amino acid, derived from the aminobutyl moiety
of spermidine, represents an integral part of eukaryotic
initiation factor 5 A (eIF5A) [10] that is essential for
eukaryotic cell proliferation [11]. A further unique

protein, ODC antizyme, is intimately involved in the
metabolism of the polyamines. Antizyme was initially
discovered in the late 1970s as a protein inhibitor of
ODC [12–14]. Subsequently, it became obvious that the
antizyme not only inhibited ODC activity but it also
facilitated its degradation by targeting ODC to 26S
proteasome [15,16]. It also appears that the antizyme is
responsible for the feedback inhibition of polyamine
transport [17]. The regulation of ODC antizyme expres-
sion is unique as polyamines directly induce ribosomal
frameshifting in decoding antizyme resulting in the
formation of full-length functional protein [18,19].
Recently, a nuclear localization has been described for
antizyme (and ODC) during mouse development [20] and
antizyme appears to have nuclear export signals [21].
Recent studies have likewise indicated that antizyme
interacts in the nucleus with the transcription factor
Fig. 1. The metabolism of the polyamines. ODC, ornithine decarboxylase; Spd, spermidine; AdoMetDC, S-adenosylmethionine decarboxylase;
dcAdoMet, decarboxylated AdoMet; Spm, spermine; SSAT, spermidine/spermine N
1
-acetyltransferase; PAO, polyamine oxidase; SMO, spermine
oxidase; eIF5A, eukaryotic initiation factor 5 A. The superscripts indicate the genetic modification of the genes: TG, transgenic; KO, knockout;
DN, dominant negative mutation.
878 J. Ja
¨
nne et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Smad1 and with HsN3, a proteasome subunit [22]. The
complex then appears to recruit SNIP 1, the repressor of
CBP/p300 [23]. The latter apparently implies that the
antizyme has functions beyond the regulation of ODC

activity/turnover and polyamine transport.
There are several recent review articles dealing in more
detail with polyamine metabolism, their putative functions
and polyamine-related pharmacological/clinical approaches
[24–27].
Putative cellular functions of the polyamines
Polyamines are organic cations that are positively charged
under physiological conditions. Thus, they are expected to
interact with negatively charged molecules, such as nucleic
acids, phospholipids, etc., within the living cells. What
makes them different from divalent cations, for instance?
There are two fundamental differences between the natural
polyamines and divalent cations, such as Mg
2+
and Ca
2+
.
(a) The positive charges in the polyamines are differentially
spaced within a flexible carbon backbone and hence
electrostatic interactions with other cellular components,
most notably with negatively charged macromolecules, in
all likelihood are more flexible than those exerted by
divalent cations. (b) As described above, the polyamines
possess extremely sophisticated metabolic machinery for the
regulation and maintenance of their intracellular homeo-
stasis. Of course, one can argue that the latter likewise
holds true for divalent cations in terms of differential
cellular compartmentalization. There are more than 1500
research reports describing effects exerted by the polyamines
in great diverse experimental systems in vitro. If not all, most

of these experiments may not be relevant as regards the true
cellular functions of the polyamines, as no one knows the
concentrations of free polyamines in a living cell. Especially
the higher polyamines, spermidine and spermine while
present in millimolar concentrations in the cell are expected
to be tightly bound to negatively charged cellular compo-
nents and structures leaving only an extremely tiny fraction
of them reactive. Thus, to conclude anything about the
functions of the polyamines based on biological effects
exerted by them in vitro is a hopeless task without any solid
ground.
As regards the physiological functions of the polyamines,
however, experimental evidence exists that assigns specific
roles to the polyamines in general and to individual
polyamines in particular. The polyamines, especially sper-
midine and spermine, interact with DNA with reasonable
specificity. In fact, they can alter the structure of DNA, such
as B to Z conversion, and are thus likely to affect the
function of DNA [24]. An example of an extremely specific
interaction between the higher polyamines and polynucleo-
tides is the ribosomal frameshifting induced by the poly-
amines in decoding of ODC antizyme [18]. Polyamines
appear to have an indispensable role in cell proliferation, as
specific inhibition of their biosynthesis invariably halts
the growth of mammalian cells. This likewise applies to
polyamine depletion achieved by an activation of their
catabolism. The biosynthetic and catabolic enzymes of the
polyamines have become a meaningful target for cancer
chemotherapy. However, the impressive results obtained in
cell cultures and even with tumor-bearing animal models

have not fully translated to clinical practice apparently due
to the sophisticated compensatory mechanisms aimed at
maintaining polyamine homeostasis [25,26]. In fact, poly-
amines may have a dual role in cellular functions by
promoting cell growth or inducing apoptosis when they
occur in excess [24]. An inhibition of polyamine biosynthesis
has met much greater success in the treatment of certain
parasitic diseases, such as African sleeping sickness, under
clinical conditions [24]. A further function of the poly-
amines, specific to spermidine, is the formation of hypusine,
an integral component of the eIF5A [10]. As the latter factor
is essential for the proliferation of mammalian cells [11], it
is often difficult to judge whether spermidine depletion-
induced growth arrest is attributable to the polyamine itself
or whether it is related to the shortage of hypusine to form a
functional translation initiation factor. Finally, the poly-
amines specifically interact with certain ion channels, such as
N-methyl-
D
-aspartate receptor, inward rectifying potassium
channels and voltage-dependent Ca
2+
channels [24]. As
these interactions occur at such low (nanomolar) concen-
trations, it is highly likely that they are relevant also under
conditions in vivo.
Genetic engineering has been directed to almost every
single reaction of polyamine metabolism in transgenic
animals and embryonic stem cells. Individual enzymes
include arginase II, ODC, antizyme, AdoMetDC, spermi-

dine synthase, spermine synthase and SSAT the genes of
which have been either generally or tissue-specifically
overexpressed or disrupted. The following sections will
describe in detail the consequences of activated polyamine
biosynthesis, activated polyamine catabolism, antizyme
overexpression and disruption of individual enzyme genes.
A short recent review also covers some of the transgenic
mouse models [28].
Activation of polyamine biosynthesis
in transgenic rodents
Overexpression of
ornithine decarboxylase
Polyamine homeostasis. The first animal with genetically
engineered polyamine metabolism was a transgenic mouse
overexpressing the human ODC gene under its own
promoter [29]. The transgene was overexpressed practically
in all tissues of the transgenic mice in a position-independent
and gene copy number-dependent fashion [30]. While the
parenchymal tissues displayed moderately elevated ODC
activity, some tissues, such as testis and brain, showed an
enzyme activity that was 20–80 times higher than that in the
respective tissues of their nontransgenic littermates. The
high activity of ODC greatly expanded tissue pools of
putrescine, especially in testis and brain [31], but, with the
possible exception of testis, was not reflected in any
alterations of tissue levels of spermidine and spermine [31].
The fact that testis and brain showed the most expanded
putrescine pools in the transgenic animals may be attribut-
able to their less permeable blood/tissue barriers than in
other tissues. This apparent block between putrescine and

the higher polyamines is especially puzzling as analyses of
SSAT and PAO activities and urinary content of the
polyamines gave no signs indicative of an activation
of polyamine catabolism [31] or their enhanced urinary
Ó FEBS 2004 Genetic approaches to polyamine functions (Eur. J. Biochem. 271) 879
excretion [32] in the transgenic animals. An elevation of
tissue contents of
L
-ornithine through the inhibition of
ornithine transaminase only further expanded putrescine
pools but not those of spermidine or spermine [32],
indicating that the tissue ornithine pool became rate-limiting
under these conditions. These results led us to conclude that
one of the major functions of the polyamine homeostatic
system in nondividing (or nonstressed) mammalian cells is
to prevent an excessive accumulation of the higher poly-
amines spermidine and spermine, in fact, a view already
expressed by Davis et al. [33] in their review article. If
correct, this would require some sort of physical or chemical
sequestration of putrescine under these conditions.
Spermatogenesis. While establishing the first transgenic
ODC overexpressing transgenic mouse line (UKU2), we
generated a transgenic founder male displaying extremely
high testicular ODC activity. This male turned out to be
infertile and subsequent histological examination of testes
revealed greatly reduced amount of germinal epithelium
and total absence of ongoing spermatogenesis resembling a
syndrome causing infertility in man and known as ÔSertoli
cells-only syndromeÕ [29]. A closer examination of the
members of the UKU2 line revealed a significant decrease

also in their sperm count [29]. Our subsequent studies
indicated that an enhanced testicular ODC activity has
a dual effect on spermatogenesis; a moderately enhanced
putrescine accumulation stimulates mitotic DNA synthesis
while excessive accumulation of the diamine inhibits meiotic
DNA synthesis, particularly upon advancing of age [34],
ultimately leading to male infertility [29]. Highly relevant to
these findings is a recent discovery of a testis germ cell-
specific ODC antizyme (antizyme 3) [35]. The expression of
this antizyme is strictly restricted to testis and it is expressed
stage-specifically in postmeiotic germ cells, i.e. during late
spermatogenesis [35]. As indicated by our results with ODC
transgenics, excessive putrescine accumulation during late
spermatogenesis appears to be detrimental to the germ cells
and, hence, antizyme 3 apparently functions to limit any
ODC expression to the early spermatogenesis. The trans-
gene-derived ODC seemingly ÔoverwhelmsÕ the normally
occurring antizyme and leads to an excessive accumulation
of putrescine in late-stage germ cells [35,36].
Spontaneous tumorigenesis. Elevated ODC activity and
expanded pools of the polyamines are commonly associated
with tumorigenesis and a role of an oncogene-like protein
has been assigned to ODC [26,37]. Under cell culture
conditions, forced expression of ODC appears to result in
malignant transformation [37,38] and cells overproducing
ODC are able to form tumors in nude mice [39]. We
subjected the ODC-overexpressing transgenic mice to a
long-term survival study in order to assess whether very
high constitutive tissue ODC activity would predispose the
animals to an enhanced general tumorigenesis. At two years

of age, these animals still displayed 20–50 times higher tissue
ODC activity than their syngenic littermates, yet macro-
scopic and microscopic examination of organs did not
reveal any difference between syngenic and transgenic mice
as regards spontaneous tumor incidence [40]. These findings
are supported indirectly by results obtained with growth
hormone overexpressing transgenic mice, which, in addition
to constitutively elevated circulating growth hormone levels,
showed enhanced ODC activity in liver and some other
tissues but did not display any signs of malignant transfor-
mation even at advanced age [41]. A more recent study with
transgenic mice carrying mammary tumor virus long-
terminal repeat-driven ODC cDNA suggests an increased
incidence of spontaneous tumors in the transgenic animals
[42]. The tumors included mammary carcinomas, intestinal
adenocarcinoma and a vascular liver tumor, however, a
direct connection to ODC is questionable as the listed
transgenic tissues displayed lower ODC activity than the
corresponding tissues of syngenic mice. Another strange
feature is the fact that among 17 nontransgenic animals, no
pathological findings were observed at 2 years of age, which
is close to the end of the life-span of a mouse [42]. According
to a recent report, spontaneous tumor incidence in old mice
of CH3 background (used in the cited study) is 40% [43].
Skin. Transgenic mice overexpressing human ODC under its
own promoter did not show any macroscopic or microscopic
skin abnormalities, yet these animals appeared to be more
sensitive than their syngenic littermates to developing skin
papillomas in response to the two-stage chemical (initiation
and promotion) skin tumorigenesis [44]. Targeted (using

bovine keratin promoter) overexpression of truncated ODC
in the skin of transgenic mice caused a number of phenotypic
abnormalities including early and permanent loss of hair,
excessive skin wrinkling, development of dermal follicular
cysts, enhanced nail growth and spontaneous tumor devel-
opment [45]. The fact that hair loss was causally related
to ODC overexpression and putrescine accumulation was
convincingly proved by experiments showing that early
administration of 2-difluoromethylornithine (DFMO), an
irreversible inhibitor of ODC [46], prevented the hair loss and
partially normalized skin histology [47]. The same authors
likewise showed that hair follicle-targeted overexpression of
ODC not only predisposed the transgenic animals to skin
tumorigenesis but the tumors developed in response to a
single carcinogen application without a subsequent tumor
promotion [48]. The use of DFMO reversibly blocked the
formation of squamous papillomas in the transgenic animals,
which led the authors to conclude that polyamines, most
notably putrescine, control the development and mainten-
ance of neoplastic phenotype [49]. The overexpression of
ODC appears to co-operate with v-Ha-ras oncogene as
doubly transgenic mice carrying both keratin promoter-
driven ODC transgene and v-Ha-ras developed spontaneous
skin tumors unlike the singly transgenic animals [50]. In
addition to chemical carcinogenesis, transgenic mice over-
expressing ODC under the control of keratin promoter were
also sensitized to photocarcinogenesis as indicated by the
more rapid development of skin tumors in these animals in
comparison with their syngenic littermates [51]. As in the case
of chemical carcinogenesis, the development of tumors in

response to the ultraviolet radiation was completely preven-
ted by DFMO [51].
The role of ODC in skin tumorigenesis has also been
approached by generating transgenic mouse line expressing
keratin promoter-driven truncated dominant-negative ODC
mutant [52]. In spite of an inhibition of wild-type ODC
expression in short-term experiments, these animals formed
as many tumors as controls in response to the two-stage skin
880 J. Ja
¨
nne et al. (Eur. J. Biochem. 271) Ó FEBS 2004
tumorigenesis protocol. A plausible explanation for the
failure to inhibit tumorigenesis is the competition of the
mutant ODC for the binding of the antizyme and releasing
thewild-typeenzyme[52].
The mechanisms whereby ODC overexpression enhances
skin tumorigenesis are largely unknown. Two recent reports
link ODC overexpression to histone acetylation as both
histone acetyltransferase and deacetylase activities were
elevated in the skin of the ODC transgenic animals and
histones were hyperacetylated in cultured skin cells over-
expressing ODC [53,54]. Skin tumors obtained from doubly
transgenic ODC/Ras mice displayed an exceptionally high
histone acetyltransferase activity [54]. These changes were
fully reversible by DFMO treatment. These findings may
imply that elevated intracellular polyamines can influence
the chromatin organization and possibly alter specific gene
expression to promote tumor progression [54].
As further regards skin, it may be worth of mentioning
that an overexpression of arginase I in the enterocytes of

transgenic mice elicits arginine deficiency that affects skin,
muscle and lymphoid development, however, in the absence
of altered polyamine tissue pools [55].
Central nervous system. The role of the polyamines in
normal and pathological brain is not only under active
research but the existing views of their roles are highly
conflicting. A vast number of experiments have shown that
brain insults, either physical or chemical, inevitably activate
the biosynthesis of the polyamines through an induction of
ODC and concomitant accumulation of putrescine. This
phenomenon is mostly understood in terms that the
induction of ODC and the enhanced accumulation of
putrescine is causally related to the neuronal damage rather
than representing an adaptive response [56,57]. Spermidine
and spermine interact as agonists with the N-methyl-
D
-aspartate (NMDA) receptor [58,59], a prolonged activa-
tion of which could be responsible for neuronal damage [56].
Unlike the higher polyamines, putrescine is believed to act
as a weak antagonist of the NMDA receptor [59]. The
experiments with transgenic mice and rats overexpressing
ODC have generated new information strongly suggesting
that an enhanced accumulation of putrescine in brain is a
neuroprotective measure rather than a cause of neuronal
damage.
As indicated earlier, transgenic mice overexpressing ODC
show the greatest expansion of the putrescine pool in brain
and testis. In the long-term survival experiment, we
examined the transgenic animals and their syngenic litter-
mates at 2 years of age and found no macroscopic or

microscopic signs of neuronal degeneration in the transgenic
brains [40]. This means that life-long constitutive over-
expression of ODC and enhanced accumulation of putres-
cine in the brain is tolerated with no harmful consequences.
Consequent experiments with the ODC overexpressing
mice indicated that these animals showed a significantly
elevated seizure threshold to both chemical (pentylene-
tetrazol) and physical (electroshock) stimuli and impaired
performance in spatial learning and memory tests. The
elevated seizure threshold was not due to any changes in the
brain levels of the two major neurotransmitter amino acids,
glutamate and c-aminobutyric acid [60]. Mg
2+
is a well
known voltage-dependent, physiological blocker of the
glutamate-mediated excitatory currents inhibiting ionic
conductance through the NMDA channel [61]. Thus,
elevated free Mg
2+
could potentially block the NMDA
receptor, yet our studies revealed that free Mg
2+
was
significantly lowered (40%) in the brain of transgenic
animals [62]. Taken together, these results suggest that
endogenous putrescine may play a physiologically relevant
role at the NMDA receptor as these receptors have a well
documented role in the induction of seizure activity [63] and
mediating spatial encoding [64]. The finding indicating that
the transcript levels of several neurotrophins were elevated

in the brain of the transgenic animals may likewise
contribute to the apparent neuroprotection [65].
The view that elevated brain putrescine offers neuropro-
tection, or at least is not neurotoxic, was supported by a
series of studies where ODC overexpressing transgenic mice
and rats were subjected to cerebral ischemia. Transgenic
mice suffering from incomplete forebrain ischemia due to
the occlusion of common carotid arteries did not show any
signs indicative of putrescine neurotoxicity as judged by
changes of energy metabolism (assessed with the aid of
nuclear magnetic resonance spectroscopy), induction of
immediate early genes and the extent of hippocampal
necroses [66]. Similar results were obtained with a transgenic
rat model for ODC overexpression after permanent middle
cerebral artery occlusion [67]. A comparison of ODC
overexpressing transgenic rats with syngenic and DFMO-
treated rats after transient middle cerebral occlusion indi-
cated that the ischemia-reperfusion damage developed more
slowly and the infarct volumes were smaller in the trans-
genic animals [68,69]. These studies clearly indicate that an
induction of ODC and the concomitant accumulation of
brain putrescine are adaptive responses to noxious insults
and do not enhance the lesion development.
Cardiac hypertrophy. Agents that cause cardiac hyper-
trophy are known to activate polyamine biosynthesis and
elevate their cardiac levels [70,71]. Although cardiac hyper-
trophy in response to b-adrenergic agonists can be prevented
by a specific inhibition of ODC by DFMO [71,72], it is by no
means clear whether an enhanced polyamine biosynthesis
and accumulation per se can cause cardiac hypertrophy.

This issue was addressed by generating transgenic mice
with targeted overexpression of ODC in the heart. Using
truncated ODC driven by a-myosin-heavy-chain promoter,
a more than 1000-fold overexpression was achieved [73].
Cardiac putrescine pool was expanded by a factor of 50 and
that of spermidine by a factor of four while spermine content
was only slightly elevated [73]. The enormous ODC activity
apparently depleted tissue ornithine, as substantial amounts
of lysine-derived cadaverine accumulated in the heart. Even
though the altered polyamine pools in transgenic animals
did not lead to hypertrophic phenotype, this condition
co-operated with b-adrenergic stimulation resulting in severe,
sometimes fatal, cardiac hypertrophy compared with only
mild hypertrophy in the similarly treated nontransgenic
littermates [73]. It is noteworthy that in this study, ODC
overexpression resulted in substantial expansion of tissue
spermidine pool too that is not often observed in other ODC
overexpressing transgenic models.
Polyamines are known to be modulators of inward
rectifying K channels (K
ir
channels) spermine being 100-fold
Ó FEBS 2004 Genetic approaches to polyamine functions (Eur. J. Biochem. 271) 881
and spermidine 10-fold more potent blockers of the
channels than putrescine or Mg
2+
[74–76]. Studies on
inward rectification properties of cardiomyocytes isolated
from transgenic mice with heart-targeted ODC overexpres-
sion, unexpectedly revealed that, in spite of massive over-

accumulation of putrescine and cadaverine, the rectification
properties were essentially unaltered [76]. Two explanations
for this finding were offered: (a) these diamines did not
significantly contribute to the rectification or (b) their free
concentrations were not altered despite the massive rise in
total levels. Interestingly, the authors reached the conclusion
proposing that most of the putrescine (and cadaverine) is
not free but is sequestered within the cell [76]. If correct, this
would also explain the commonly observed biosynthetic
block from putrescine to spermidine under conditions of
massive putrescine over-accumulation.
In order to exploit whether the tissue concentrations of
spermidine and spermine could be increased, we generated
several transgenic mouse lines overexpressing rat Ado-
MetDC gene. Among the five lines produced, none
displayed an increase in AdoMetDC activity nearly as
dramatic as seen in ODC overexpressing animals. The
increase in the enzyme activity was at the best fivefold in
comparison with their syngenic littermates and the tissue
pools of spermidine and spermine of transgenic animals
showed only marginal changes [77]. Also, in hybrid mice
overexpressing both ODC and AdoMetDC, the tissue levels
of the higher polyamines did not differ from those in
syngenic mice. Pulse labeling experiments with primary fetal
fibroblasts obtained from doubly transgenic mice indicated
that polyamine flow was faster in the transgenic than in
nontransgenic fibroblasts. AdoMetDC overexpressing ani-
mal did not show any phenotypic alterations [77].
One transgenic mouse line likewise generated moderately
(two–sixfold) overexpressing human spermidine synthase

gene but showing no perturbations in tissue polyamine
homeostasis or phenotypic changes [78]. With combined
overexpression of ODC and spermidine synthase in hybrid
transgenic mice neither brought about any changes in
normal polyamine homeostasis [78].
Activation of polyamine catabolism
Overexpression of
spermidine/spermine
N
1
-acetyltransferase
Polyamine homeostasis. As indicated earlier (Fig. 1), sper-
midine and spermine are converted back to putrescine
through the concerted action of SSAT and PAO. PAO is
constitutively expressed and strongly prefers acetylated
polyamines as the substrates [3] while SSAT is highly
inducible, has a very short half-life and serves as the rate-
controlling enzyme in polyamine backconversion [4]. The
first founder animal overexpressing SSAT (UKU169F
0
)
was a female mouse harboring more than 50 SSAT gene
copies in its genome. The animal was extremely small,
hairless and infertile. Tissue polyamine pools were dramat-
ically distorted. The transgenic brain contained an extremely
high concentration of N
1
-acetylspermidine, a compound
not normally found in mouse tissues and greatly reduced
spermidine pool, while in liver, the putrescine pool was

strikingly expanded and that of spermine greatly reduced
[79]. As the animal was infertile, no transgenic line could be
established. The second founder animal was male that gave
rise to two different kinds of offspring, animals harboring
only a few SSAT gene copies and retaining their hair (line
UKU165a) and animals having more than 20 SSAT gene
copies and permanently losing their hair at 3–4 weeks of age
(line UKU165b). Members of the UKU165a showed only
marginal alterations in their tissue polyamine pools while
members of the UKU165b displayed polyamine pool
changes typical for SSAT overexpression: large increase in
tissue putrescine pool, appearance of N
1
-acetylspermidine
and decreases in spermidine and/or spermine pools [79].
Interestingly, these changes developed in the presence of
only moderately elevated tissue SSAT activity [79]. Our
results (unpublished) have indicated that overexpression of
SSAT under these conditions does not decrease the hepatic
pool of acetyl-CoA.
In an attempt to correct SSAT-induced perturbations in
polyamine homeostasis, we generated a hybrid transgenic
mouse line overexpressing both ODC and SSAT under the
control of mouse metallothionein I promoter. Unexpect-
edly, these animals showed much more striking signs of
activated hepatic polyamine catabolism than the SSAT
overexpressing animals [80]. Even under the condition of
severe depletion of spermidine and spermine pools, tremen-
dously high tissue putrescine was not driven further to
replenish the reduced pool of spermidine. We understand

from these results that catabolism is the overriding control
mechanism in polyamine metabolism [80].
Regulation of transgene-derived SSAT by polyamine
analogues. SSAT is known to be powerfully induced by
the higher polyamines and especially by certain polyamine
analogues [4]. The regulation of SSAT expression by
polyamines and their analogues apparently occurs at many
levels of gene expression. These include enhanced transcrip-
tion and stabilization of the transcript [81,82], enhanced
mRNA translation [83,84] and stabilization of the enzyme
protein [85]. The transgenic animals and cells derived from
them typically accumulate large amounts of SSAT-specific
mRNA that is, however, extremely poorly translated in the
absence of polyamines or their analogues [79,86]. This is
exemplified by the observation showing that in the pre-
sence of N
1
,N
11
-diethylnorspermine (DENSPM), a power-
ful inducer of SSAT, nontransgenic cells display SSAT
activity 10· higher than that in transgenic cells not exposed
to the analogue but containing 10· more SSAT-specific
mRNA [86]. In fact, working with transgenic mice overex-
pressing SSAT under the control of mouse metallothionein
I promoter, we found striking evidence for a post-tran-
scriptional regulation of the transgene expression by
DENSPM [87]. In spite of the heterologous promoter,
hepatic transgene-derived SSAT was stimulated more than
40 000-fold by the analogue with marginal changes of

transcript levels [87]. We proposed that the polyamine
analogue could directly interact with SSAT mRNA and
improve the translability of the message. It is not excluded
that polyamine analogues could alter the splicing of SSAT
pre-mRNA as certain viruses appear to induce alternative
splicing of the SSAT transcript [88].
Polyamine analogues are potential cancer chemothera-
peutic agents and, in fact, DENSPM has undergone clinical
882 J. Ja
¨
nne et al. (Eur. J. Biochem. 271) Ó FEBS 2004
trials [89]. The antiproliferative action of the polyamine
analogues is believed to be attributable to an induction of
SSAT activity and subsequent depletion of the higher
polyamines. This conclusion is based on comparisons
between the different inductions of SSAT activity by the
analogues and their antiproliferative activity [90–93]. The
interpretation of such comparisons between genetically
dissimilar cell lines may, however, be difficult as the
analogues may have multiple sites of action in different cell
lines. We approached the issue by isolating fetal fibroblasts
from nontransgenic and SSAT overexpressing mice and
exposing the cells to the analogue. We now had a pair of
similar cells differing only in the number of SSAT gene
copies. We found that the SSAT overexpressing cells were
much more sensitive to DENSPM-induced growth inhibi-
tion than the nontransgenic cells [86]. Transgenic mice
overexpressing SSAT were also more sensitive to the general
toxicity of the polyamine analogue [94]. A recent report
indicates that small interfering RNA targeted to SSAT

mRNA not only prevented SSAT induction by DENSPM,
but also prevented apoptosis [95].
As will be shown below, overexpression of SSAT in
transgenic animals not only profoundly altered tissue
polyamine homeostasis, but likewise created a very complex
phenotype affecting skin, fat depots, female fertility, pan-
creas, liver and the central nervous system.
Skin. As indicated earlier, the first founder animal
permanently lost its hair at an early age, as did the
members of the UKU165b line harboring more than 20
copies of the SSAT gene [79]. Paradoxically, the hairless
skin histology was practically identical to that found in
transgenic mice with hair follicle-targeted overexpression
of ODC [45], i.e. replacement of hair follicles by large
dermal cysts (apparently filled with keratin) and epidermal
utriculi, extensive wrinkling of the skin upon aging and
lack of subcutaneous fat depots [79,96]. Figure 2 shows a
young SSAT transgenic mouse with its syngenic littermate
(A) and an old transgenic animal displaying excessive
wrinkling of the skin that gives a ÔrhinomouseÕ appearance
to the animal (B). The lower panels in Fig. 2 depict the
histology of normal skin (C), skin of young (D) and an
old (E) transgenic mouse. Note that the normal hair
follicles (C) are replaced by dermal cysts (D), which
become extremely large in old animals (E).
In case of ODC overexpression, the hair loss was
attributable to an excessive accumulation of putrescine in
the skin, as the loss of hair could be prevented by an
early administration of the ODC inhibitor, DFMO [47].
Although indirectly proved, over-accumulation of putres-

cine is in all likelihood responsible for the hair loss also
observed in SSAT overexpressing mice. This view is
supported by experimental findings indicating that putre-
scine was constitutively over-accumulated in the skin of
these animals and, while the animals properly completed
their first hair-cycle, they failed to commence the second
anagen phase due to lack of functional hair follicles.
Moreover, doubly transgenic mice overexpressing both
SSAT and ODC with extremely high levels of putre-
scine in the skin displayed distinctly more severe skin
histology (significantly larger size of the dermal cysts)
than did the singly transgenic mice [96]. Transgenic
mice overexpressing SSAT under the control of mouse
metallothionein I promoter also lost their hair but much
later than those overexpressing the gene under its own
promoter [87].
When subjected to the two-stage skin tumorigenesis
protocol, SSAT overexpressing mice developed significantly
Fig. 2. Hairless phenotype of the SSAT overexpressing mouse. Young SSAT overexpressing mouse with its syngenic littermate (A). An old SSAT
transgenic mouse (B). Histology of normal skin showing intact hair follicles (C). Hair follicles are replaced by dermal cysts in SSAT transgenic
mouse (D). In old transgenic mouse, the cysts become larger (E) wrinkling the skin.
Ó FEBS 2004 Genetic approaches to polyamine functions (Eur. J. Biochem. 271) 883
fewer papillomas than their syngenic littermates [96]. This
may be related to the fact that in the syngenic animals, both
ODC activity and skin spermidine level increased much
more in response to the tumor promoter than in transgenic
mice [96]. Coleman et al. [97] employed another approach
to study the role SSAT in skin pathophysiology producing,
in fact, opposite results. They generated transgenic mice
expressing SSAT cDNA under bovine keratin 6 promoter,

which directs the expression to the keratinocytes of the hair
follicle. The animals were phenotypically indistinguishable
from their normal littermates and showed normal hair-
cycle. The latter may be attributable to the fact that the low
SSAT activity of skin extracts was not increased in the
transgenic animals [97]. These animals, however, were much
more sensitive to two-stage skin tumorigenesis, as judged by
tumor incidence and multiplicity, than their syngenic
littermates and showed distinctly enhanced SSAT activity
and increased putrescine and N
1
-acetylspermidine level in
the papillomas. Interestingly, cysts, derived from dilated
hair follicles, were found in the vicinity of the papillomas but
less abundantly elsewhere [97]. The obvious inconsistency
between present [97] and previous observations [96] may be
relatedtothedifferentlevelsofSSAT expression, genetic
background or hairlessness. However, mice used in our
studies [96] were of a BalbC · DBA/2 background. Mice of
DBA/2 are reportedly more sensitive to tumor promotion
than animals from a C57BL/6 background [97]. Moreover,
all the existing experimental data seem to indicate that an
activation of polyamine catabolism is more closely related
toantiproliferativeactionthantogrowthpromotion.
Female reproductive organs. Histopathological examina-
tion of the SSAT overexpressing mice revealed that of 18
tissues and organs examined only skin and female repro-
ductive tract were affected in the transgenic animals. Female
members of the transgenic line UKU165b were infertile,
their uteri were hypoplastic due to a thinner muscular layer

and stromal and glandular development was greatly
reduced. Examination of the ovaries revealed the presence
of primary and small secondary follicles, but absence of
larger developing follicles and corpus luteum [79]. Differ-
ential display analysis of a gene expression profile of uterus
and ovary indicated that the expression of lipoprotein lipase
and glyceraldehyde-3-phosphate dehydrogenase was eleva-
ted in transgenic animals [98]. Both enzymes are involved in
energy metabolism and may have detrimental effects on
myometrium and cell viability when overexpressed [98].
SSAT overexpression was also associated with induced
expression of insulin-like growth factor binding protein-2 in
the uterus and ovary and decreased expression of insulin-
like growth factor binding protein-3 in the uterus. These
changes may also contribute to the uterine hypoplasia and
ovarian hypofunction [98]. It is interesting to note, that
ODC overexpression leads to male infertility while SSAT
overexpression results in female infertility.
Pancreas. Pancreas is the richest source of spermidine in
the mammalian body and displays the highest molar ratio of
spermidine to spermine, nearly 10 [99,100]. The exact
function of such a high spermidine concentration in the
pancreas is not known, but may be related to the intense
protein synthesis that occurs in this organ. Pancreatic
growth appears to be dependent on polyamine biosynthesis,
as DFMO retards the growth of the pancreas [101], but does
not inhibit the secretory function of the exocrine part of the
organ [102]. The cellular functions of the polyamines in the
pancreas were approached by generating transgenic rat lines
overexpressing SSAT under the control of heavy metal-

inducible metallothionein I promoter [103]. Although
transgenic pancreas displayed all the signs of activated
polyamine catabolism, such as massive accumulation of
putrescine and appearance N
1
-acetylspermidine, the levels
of the higher polyamines were relatively well maintained.
Zinc induction of the promoter resulted in a striking
stimulation of the pancreatic SSAT activity in the transgenic
animals, but not in the syngenic animals, that was
accompanied by an almost total depletion of pancreatic
spermidine and spermine and development of histologically
verified acute necrotizing pancreatitis [103]. The possibility
that pancreatitis would have been caused by reactive oxygen
species generated by the action of PAO was excluded by
experiments in which PAO was specifically inhibited before
zinc administration, showing that the latter inhibition did
not alleviate, but rather worsened the pancreatitis [103]. A
further piece of evidence causally relating the development
of pancreatitis to the profoundly depleted pancreatic
polyamine pools came from experiments showing that the
inflammatory process could be totally prevented, as judged
by histopathology and plasma a-amylase activity, by a prior
administration of a-methylspermidine, a metabolically
stable spermidine derivative [104]. The results indicated that
the higher polyamines are required for the maintenance of
metabolic and structural integrity of the pancreas. Induction
of SSAT as a cause of acute pancreatic inflammation may
have wider applications, especially concerning drug-induced
pancreatitis. Gossypol, a cotton seed-derived male antifer-

tility agent [105], is known to induce SSAT expression in
canine prostate cells [106]. We recently showed that the drug
activates polyamine catabolism in the pancreas of normal
rats and induces acute pancreatitis through a profound
depletion of polyamine in transgenic rats overexpressing
SSAT [107]. It thus appears meaningful to screen drugs
known to induce pancreatitis for their effect on pancreatic
polyamine catabolism.
Liver. Polyamines are intimately associated with the growth
of mammalian cells. One of the first animal models
demonstrating this involved regenerating rat liver after
partial hepatectomy. Partial hepatectomy is known to cause
an early induction of ODC in the regenerating liver remnant
[108,109] followed by a sequential accumulation of putres-
cine and spermidine with a slight decrease in spermine [110].
Even though attempts have been made to pharmacologi-
cally inhibit rat liver regeneration through blocking ODC,
the results have been conflicting [111,112]. Partial hepatec-
tomy of transgenic rats expressing metallothionein promo-
ter-driven SSAT dramatically induced the enzyme at 24 h
after the operation that consequently depleted the hepatic
spermidine pool by 80%. As judged by a number of
proliferation indicators, the transgenic rats failed to initiate
liver regeneration in striking contrast to their syngenic
littermates [113]. Only when hepatic spermidine concentra-
tion was increased to the preoperative level (apparently due
to very high ODC activity) at day 3 after the operation, liver
884 J. Ja
¨
nne et al. (Eur. J. Biochem. 271) Ó FEBS 2004

regeneration slowly commenced in the transgenic animals
[113]. The view that the delayed initiation of liver regener-
ation in the transgenic animals was causally related to the
depletion of hepatic spermidine and spermine pools was
strongly supported by experiments revealing that a-methyl-
spermidine given prior to the partial hepatectomy fully
restored the regeneration [104].
As indicated earlier, SSAT overexpressing mice were
extremely sensitive to the toxic effects exerted by polyamine
analogues. Treatment of transgenic mice overexpressing
SSAT under the control of metallothionein promoter with
DENSPM effectively depleted hepatic polyamine pools and
resulted in marked mortality that was associated with
ultrastructural changes in liver, most notably mitochondrial
swelling [87].
Central nervous system. In comparison with the ODC
transgenic mice, overexpression of SSAT resulted in even
greater expansion of putrescine pool in different regions of
the brain. In situ hybridization analyses of the transgenic
mice indicated that SSAT was overexpressed in all brain
tissue [114]. Some experimental work appears to link
enhanced SSAT activity to neuronal damage. This view is
based on the finding indicating that kainate-induced seizure
activity resulted in an early stimulation of SSAT activity in
rat brain [115]. Neurotoxicity of kainate is mediated by a
Ca
2+
-dependent process and the drug is particularly toxic to
pyramidal cells of hippocampal and cortical neurons [116].
Overexpression of SSAT appears to protect the transgenic

animals from kainate-induced toxicity. This was manifested
as a substantially reduced (50%) overall mortality of the
transgenic mice, in comparison with their syngenic litter-
mates, in response to high-dose kainate [114]. The trans-
genicity likewise offered a distinct neuroprotection exhibited
as a reduced expression of glial fibrillary acidic protein, an
commonly used marker of neuronal injury, and no loss of
hippocampal neurons in response to kainate in the
transgenic animals in comparison with wild-type mice
[114]. These results support our earlier view suggesting that
expanded pools of brain putrescine, irrespective whether
derived from ODC or SSAT overexpression, have a distinct
neuroprotective role.
The SSAT overexpressing mice likewise showed a
significantly elevated threshold, in comparison with their
syngenic littermates, to pentylenetetrazol-induced seizure
activity involving both tonic and clonic convulsions [117].
Although pentylenetetrazol principally induces epilepsy-like
seizure activity through the inhibition of c-aminobutyric
acid (GABA), the major inhibitory neurotransmitter [118], a
number of reports indicate that antagonists of the NMDA
receptor elevate the seizure threshold to pentylentetrazol
[119,120]. Interestingly, the difference of seizure threshold
to pentylenetetrazol between the transgenic and wild-type
animals totally disappeared when ifenprodil, a known
NMDA receptor antagonist acting at the polyamine site
of the receptor [121,122], was administered prior to
pentylenetetrazol [117]. In addition to the elevated seizure
threshold, SSAT overexpression likewise protected the
animals from pentylenetetrazol-induced neuron loss in

the hippocampus [117]. These results are clearly in line with
the notion that grossly elevated putrescine levels or the
greatly increased (up to 40-fold) molar ratio of putrescine to
the higher polyamines in the transgenic brain creates a
partial NMDA receptor blockade [117].
Transgenic expression of ODC antizyme
As mentioned earlier, antizyme not only inhibits ODC
activity, but also facilitates proteasomal degradation of
ODC protein and represses polyamine transport. In fact,
antizyme occurs in at least three isoforms (antizyme1–3)
[16,123]. Unlike antizyme 1, which both inhibits ODC
activity and facilitates the degradation of the enzyme
protein, antizyme 2 appears to lack the latter function
[16]. As mentioned earlier, antizyme 3 is only expressed in
testis during late spermatogenesis [35]. Antizyme was shown
to be translocated into nucleus during embryonic develop-
ment [20] and the protein contains two independent nuclear
export signals [21]. Moreover, antizyme forms a ternary
complex with the transcription factor Smad1 and protea-
somal subunit HsN3 that is translocated into nucleus in
response to bone morphogenetic protein receptor activation
[22]. In the nucleus, this complex further recruits CBP/p300
repressor SNIP1 and is degraded [23]. A recent review [123]
also suggests, based on so far unpublished report, that cyclin
D1 and its associated kinase cdk4 interact with antizyme
and are degraded in proteasome in a antizyme-dependent
fashion. These findings may indicate that ODC antizyme is
a general targeting protein for proteasomal degradation.
Some recent observations likewise suggest that antizyme
possesses functions unrelated to the polyamine metabolism.

Antizyme expression is up-regulated in melanoma cells
in response to interleukin-1 [124] and antizyme levels are
reduced in certain experimental cancers [125]. Antizyme
seems to play a specific role in mammalian prostate.
Spermine has been identified as an endogenous growth
inhibitor in human prostate [126] and it inhibits the growth
of poorly metastatic, but not of highly metastatic, rat
prostate carcinoma cells [127]. The failure of spermine to
inhibit the latter cells is believed to be attributable to the
inability of the polyamine to induce antizyme in the highly
metastatic cells [127]. If antizyme has functions beyond the
metabolism of the polyamines, especially if it interacts with
the key players of the cell cycle control, caution should be
exercised in interpreting experimental results showing
antizyme-dependent growth inhibition.
Targeted antizyme 1 expression has been achieved in
several transgenic mouse models. The structural part of
transgene construct used has been a mutated rat antizyme
cDNA where a single nucleotide deletion eliminates the
need for frameshifting in translation [128].
Cardiac hypertrophy
Two transgenic mouse lines were generated constitutively
overexpressing mutated antizyme cDNA under the control
of cardiac a-myosin heavy chain promoter targeting the
expression to the heart [128]. Even though antizyme
effectively inhibited cardiac ODC activity, some residual
activity was left and the changes in polyamine pools
were small with no changes in cardiac function [128].
A prolonged exposure of syngenic mice to isoprenaline
elevated cardiac ODC activity, significantly expanded

putrescine and spermidine pools and increased cardiac
Ó FEBS 2004 Genetic approaches to polyamine functions (Eur. J. Biochem. 271) 885
growth. Identical treatment of transgenic mice did not
activate cardiac polyamine biosynthesis and their tissue
accumulation, but induced similar cardiac hypertrophy as
seen in wild-type mice [128]. The result was somewhat
unexpected as earlier studies have indicated that a specific
inhibition of ODC by DFMO can prevent b-adrenergic
agonist-induced cardiac hypertrophy [72,129].
Skin tumorigenesis
Antizyme expressionhasalsobeentargetedintoskinwith
bovine keratin 5 and keratin 6 promoters in several lines of
transgenic mice in order to study the role of ODC in the
two-stage skin tumorigenesis. In comparison with syngenic
mice, the transgenic mice displayed greatly reduced
epidermal and dermal ODC activity and spermidine
content in response to tumor promotion [130]. All the
transgenic lines showed decreased susceptibility to develop
papillomas in response to the two-stage chemical carcin-
ogenesis protocol [130]. Although earlier studies have
convincingly shown that a specific inhibition of ODC by
DFMO inhibits skin tumorigenesis in this model [131,132],
the present approach is more specific as the inhibition of
ODC activity by the antizyme occurs in skin cells and not
all over the body.
Gastrointestinal carcinogenesis
Chemical carcinogenesis in the fore-stomach of zinc-defici-
ent mice is another model where the role of ODC has been
studied using targeted expression of the antizyme. Antizyme
expression significantly reduced both tumor incidence and

multiplicity in response to N-nitrosomethylbenzylamine,
promoted apoptosis and reduced the expression of cyclin
D1 and cdk4 in the fore-stomach of the transgenic mice
[133]. The view that the reduced tumor incidence was related
to ODC inhibition and not to direct effects of antizyme on
some cell cycle regulators [123], was strongly supported by
experiments indicating that inhibition of ODC by DFMO
reduced tumor incidence and promoted apoptosis in a
similar fashion as did transgenic expression of antizyme
[133]. Based on these and the skin tumorigenesis studies
[130], the authors propose that antizyme may represent a
tumor suppressor gene.
Gene disruption technology applied to the
enzymes of polyamine metabolism
Many of the genes of the polyamine metabolizing enzymes
have been disrupted either in transgenic mice or mouse
embryonic stem cells. In addition to targeted disruption of
the genes, there is a X-linked dominant mutation in mice that
involves a genomic deletion containing spermine synthase
gene. The following paragraphs list the existing knowledge of
disruption of genes involved in polyamine metabolism.
Arginase II
Arginase enzyme, degrading arginine to ornithine and urea,
occurs in two isoforms, cytosolic arginase I, which partici-
pates in the urea cycle, and mitochondrial arginase II, which
apparently is involved in the polyamine synthesis [134].
Mice with a targeted disruption of arginase II gene have
been created recently. Homozygous arginase II-deficient
mice were viable and otherwise indistinguishable from wild-
type mice, except for showing significantly elevated plasma

arginine levels. Polyamine analyses of several tissues (brain,
liver, kidney and testis) did not reveal any differences
between mutant mice and their wild-type counterparts [135].
Although the deficiency in arginase II appears to be a
benign trait under normal conditions, it is possible that this
deficiency could be deleterious under certain pathophysio-
logical conditions.
ODC
Studies employing inhibitors of polyamine biosynthesis
have indicated that an inhibition of ODC will arrest
murine embryonic development at the morula-blastocyst
stage and an inhibition of AdoMetDC at an even earlier
stage [136]. Moreover, DFMO induces resorption of
murine embryos when given just after the first week of
gestation [137,138]. Studies with mice harboring a disrup-
ted ODC gene have revealed that heterozygous animals
were viable and fertile while homozygous embryos
underwent implantation and induced maternal deciduali-
zation, but failed to develop further. This was apparently
due to marked apoptosis occurring in the pluripotent cells
of the inner cell mass shown as substantial DNA breakage
[139]. The fact that ODC-deficient embryos developed to
the blastocyst stage, i.e., to more advanced stage than
those grown in vitro in the presence of DFMO [136], was
in all likelihood attributable to maternal components
[139]. Attempts to rescue the embryos through supple-
mentation of the pregnant females with putrescine were
unsuccessful, apparently due to the toxicity of the diamine
(high diamine oxidase activity). As to the reasons for
lethality of ODC-deficient embryos, the latter authors

offer two possibilities: oxidative DNA damage in the
absence of polyamines and inhibition of DNA methyla-
tion due to excessive accumulation of decarboxylated
AdoMet in the absence of putrescine [139].
Similar ODC gene disruption in the nematode Caenor-
habditis elegans results in a virtually normal phenotype
when grown in complex medium [140], but when the ODC-
deficient nematodes were transferred into polyamine-free
medium they showed a phenotype strongly affecting
oogenesis and embryogenesis [141].
AdoMetDC
As in the case of ODC, homozygous AdoMetDC deficiency
is not compatible with murine embryogenesis while hetero-
zygous animals were viable, normal and fertile [142].
AdoMetDC-deficient embryos developed normally to the
blastocyst stage, but died shortly thereafter or during the
early stage of gastrulation at the latest. They developed
distinctly further than did embryos cultured in the presence
of an inhibitor of AdoMetDC, methylglyoxal bis(guanyl-
hydrazone) [136]. When cultured in vitro, AdoMetDC-defi-
cient blastocysts showed an absolute growth requirement
for spermidine [142]. Unlike ODC-deficient blastocysts
[139], AdoMetDC deficiency did not result in DNA
fragmentation at the blastocyst stage [142]. The mouse
886 J. Ja
¨
nne et al. (Eur. J. Biochem. 271) Ó FEBS 2004
genome appears to also harbor an intronless AdoMetDC
gene encoding a functional enzyme [143], but according to
the present results, its contribution to AdoMetDC activity

appears to be negligible.
It thus appears that both ODC and AdoMetDC activities
are indispensable for murine embryonic development. The
reasons for the early lethality of the decarboxylase-deficient
embryos is, however, far from clear. They may be related
directly to the polyamines – oxidative stress or inadequate
synthesis of nucleic acids and proteins in the absence of the
polyamines – or, in the case of ODC deficiency, an excessive
accumulation of decarboxylated AdoMet – inhibiting DNA
methylation and thus disruption of the programming of the
embryonal development. A further possibility, which is by
no means excluded, is the prevention of hypusination of the
eIF5A in the absence of spermidine [144]. In this context, it
would be interesting to see whether embryonic and fetal
development of the decarboxylase-deficient embryos could
be rescued in vivo, not with the natural polyamines, but with
their metabolically stable analogues [104].
Spermine synthase
Targeted disruption of the spermine synthase gene has been
accomplished in mouse embryonic stem cells. In the total
absence of spermine, the targeted cells grew at a rate that was
practically similar to that of the parental cells and displayed
no morphological abnormalities [145]. The latter may be
related to the fact that spermine deficiency resulted in a
compensatory increase in cellular spermidine content, in
all likelihood due to significantly enhanced ODC and
AdoMetDC activities in the targeted cells [145]. Spermine-
deficient stem cells were more sensitive to the antiprolifer-
ative actions exerted by inhibitors of polyamine biosynthesis,
such as DFMO and methylglyoxal bis(guanylhydrazone)

(MGBG), a relatively unspecific inhibitor of AdoMetDC.
The greater sensitivity to MGBG was obviously attributable
to a substantially increased (three to sevenfold) cellular
uptake of the latter compound by the targeted cells. As
MGBG is transported into cells by a carrier used by the
natural polyamines [146], it is highly likely that spermine
deficiency results in an activation of the polyamine transport
system. The targeted cells likewise showed enhanced sensi-
tivity to the growth inhibition exerted by the polyamine
analogue, DENSPM [145]. In addition to these drugs
affecting polyamine biosynthesis and/or functions, spermine
deficiency also sensitized the cells to the antiproliferative
actions of etoposide, an inhibitor of topoisomerase II
inducing single and double strand breaks in DNA [147].
The drug inhibited the growth of spermine-deficient cells
time- and dose-dependently much more effectively than that
of the parental cells [145]. The generation of gene-disrupted
mice from the targeted embryonic stem cells proves to be
problematic, as chimeric mice obtained did not transmit the
targeted allele into the germ line.
A mouse line exists, in which the spermine synthase gene
is deleted due to mutation. There are two X-linked mouse
mutations, in which so called Phex (phosphate regulating
gene) gene is partially deleted, constituting mouse models for
X-linked hypophosphatemic rickets. These mutations are
known as gyro (Gy) and hypophosphatemia (Hyp) ([148]
and references within). The spontaneous Hyp mutation
affects only the Phex gene while in the X-ray-induced Gy
mutation, the deletion extends upstream of the Phex gene
and also involves the spermine synthase gene [148]. Both

mutations cause an enhanced renal excretion of phosphate
leading to low serum phosphate levels, impaired mineraliza-
tion and growth retardation [149]. In addition, affected Gy
males are sterile and show circling behavior and inner ear
abnormalities [149]. Tissue polyamine analyses of the Gy
mice revealed a marked reduction in hepatic and pancreatic
polyamine pools. Apart from rickets, the Gy mice showed no
macroscopic or microscopic abnormalities at autopsy [148].
Gy mouse-derived fibroblasts offered a feasible model
where the role of spermine could be studied in more detail
and under defined experimental conditions. Two types of
Gy mouse-derived cells have been used: primary cultures
of skin fibroblasts isolated from young animals and cultures
of fetal fibroblasts immortalized through transfection with
SV40 small- and large-T antigens. The use of fibroblasts is
rational as the Phex gene, also mutated in these animals, is
predominantly expressed in the bone [150] and not in
fibroblasts [151]. Many of the results obtained with the Gy
mouse-derived primary fibroblasts were in agreement with
those obtained with the targeted embryonic stem cells [145].
Spermine deficiency did not affect the basic growth of the
cells, the spermidine pool was substantially expanded and
the mutant cells were more sensitive to the growth-
inhibitory action of DFMO than the wild-type cells [151].
Unexpectedly, the mutant cells were more resistant to
oxidative stress (hydrogen peroxide), but more sensitive
UV-irradiation than their wild-type counterparts [151].
Gy mouse-derived immortalized fetal fibroblasts were
used to test whether spermine is specifically required, as
suggested by cell-free models of apoptosis [152], to elicit

caspase activation. However, no difference was found
between the mutant and the wild-type cells as regards the
expression of Bcl-2 and the activation of caspases [153]. On
the other hand, spermine deficiency combined with DFMO-
induced polyamine depletion strikingly enhanced caspase
activity triggered by UV-irradiation in the mutant cells
when compared with similarly treated wild-type cells [153].
With the exception of 1,3-bis(2-chloroethyl)-N-nitrosourea,
the absence of spermine did not sensitize the immortalized
Gy fibroblasts to most conventional and experimental
cancer chemotherapeutic agents, including etoposide [154].
These mutant cells were likewise largely as sensitive as their
wild-type counterparts to various polyamine analogues,
with the exception of diethyl derivatives of spermine and
norspermine, which were more toxic against the wild-type
cells. The latter may be related to the greater induction of
SSAT activity occurring in the spermine-containing cells
[154]. Unlike the targeted embryonic stem cells [145], the
immortalized Gy fibroblasts did not show any enhanced
sensitivity to MGBG [154].
Studies with cardiac myocytes derived from Gy mice
indicated that spermine is the major inward rectifying factor
at potassium channels [76].
As in mammalian cells, spermine is apparently not
required for the growth of Saccharomyces cerevisiae [155]
while the yeast cells show an absolute requirement for
spermidine [156]. These results, obtained with the spermine-
deficient cells, were of limited use as regards an assignment
of a specific cellular role for this polyamine. They indicate
Ó FEBS 2004 Genetic approaches to polyamine functions (Eur. J. Biochem. 271) 887

that the expanded pool of spermidine compensates the lack
of spermine, i.e. these two polyamines are at least partly
exchangeable. The partly conflicting results obtained, espe-
cially regarding the sensitivity of spermine-deficient cells
to various antiproliferative agents, may be related to the
different cell lines used, i.e. embryonic stem cells vs. primary
fibroblasts or immortalized fetal fibroblasts. Moreover, the
embryonic stem cells emerged from a targeted disruption of
the spermine synthase gene while the Gy mouse-derived cells
harbor a genomic deletion extending beyond the spermine
synthase gene.
SSAT
Targeted disruption of the SSAT gene in mouse embry-
onic stem cells has little effect on polyamine homeostasis,
with the possible exception of constantly elevated spermi-
dine pool, and the growth characteristics of the cells [6].
Expectedly, the targeted cells were more resistant than the
parental cells to the antiproliferative effect exerted by a
polyamine analogue. However, the observed resistance
was not directly related to the depletion of cellular
polyamines, as even in the absence of any SSAT activity
the intracellular polyamine pools were as effectively
depleted as in wild-type cells [6]. This may indicate that
the depletion of intracellular polyamine pools by poly-
amine analogues is based on a direct replacement of the
natural polyamines from their intracellular binding sites.
In any event, these studies created the important obser-
vation indicating that SSAT activity is absolutely neces-
sary for the conversion of spermidine to putrescine, but
not for the conversion of spermine to spermidine, paving

the way to the discovery of a specific spermine oxidase.
We have recently also generated SSAT-deficient mice, the
characterization of which is underway.
Table 1 summarizes the phenotypic changes resulting
from the genetic modifications of the enzymes involved in
polyamine metabolism.
Clinical consequences of mutations in genes
involved in polyamine metabolism
There are a few examples of clinical conditions attributable
to mutations, polymorphism or rearrangements of genes
participating polyamine metabolism.
ODC
Skin-targeted constitutive overexpression of ODC leading
to the formation of hair follicle-derived dermal cysts and
permanent hair loss [45] has been considered a model for
human papular atrichia, a rare ectodermal disorder char-
acterized by irreversible loss of hair in early childhood [157].
However, it is not known whether this clinical condition is
associated with any changes in skin ODC expression. As
identical skin histopathology and permanent loss of hair is
also associated with overexpression of SSAT [96], this
condition should be linked to an overaccumulation of
putrescine in the skin irrespective whether derived either
from ODC or SSAT overexpression.
A single report, based on specimens from 15 patients,
indicates that ODC mutations are more frequently detected
in hepatomas than in normal liver. Mutations in regions rich
in proline, glutamic acid, serine and threonine (PEST
sequences), which are supposedly involved in the rapid
degradation of ODC [158], were detected only in moderately

and poorly differentiated hepatocellular carcinomas [159].
Mutations in the PEST sequences apparently lead to a
stabilization of ODC and hence enhanced enzymatic
activity.
Enhanced ODC expression, as measured by RT-PCR,
was found in human colorectal cancer tissue in 86% of
cases. Only one missense mutation located in the PEST
region was found among 50 cancer tissue samples analyzed.
It thus appears that the observed enhanced ODC expression
in tumor tissue is generally not due to point mutations in the
coding region [160].
Table 1. Summary of the phenotypic changes resulted from genetic modifications of polyamine metabolism.
Target gene Type of modification Major phenotypic changes References
Arginase II Gene disruption Elevated plasma arginine [135]
ODC General overexpression Impaired spermatogenesis; [29,34]
enhanced skin tumorigenesis; [44]
neuroprotection [60,68]
Skin-targeted overexpression Enhanced skin tumorigenesis [45,48]
Heart-targeted overexpression Proneness to cardiac hypertrophy [73]
Gene disruption Embryonic lethality [139]
AdoMetDC Gene disruption Embryonic lethality [142]
SSAT General overexpression Hairlessness; [79]
female infertility; [79,98]
resistance to skin tumorigenesis; [96]
neuroprotection [114,117]
Liver/pancreas-targeted overexpression Inducible pancreatitis; [103,104]
delayed liver regeneration [104,113]
Skin-targeted overexpression Enhanced skin tumorigenesis [97]
ODC antizyme Skin-targeted overexpression Inhibition of skin tumorigenesis [130]
Stomach-targeted overexpression Inhibition of fore-stomach carcinogenesis [133]

Spermine synthase Gene deletion Male sterility; circling behavior; inner ear abnormalities [148,149]
888 J. Ja
¨
nne et al. (Eur. J. Biochem. 271) Ó FEBS 2004
A recent colorectal adenoma recurrence trial reveals that
a single-nucleotide polymorphism in ODC gene is a
prognostic indicator, especially in combination with the
use of aspirin, for the recurrence of adenomas [161].
The single-nucleotide polymorphism (A316G) is located in
the first intron of ODC gene between two myc-binding
domains. Adenomatous polyposis coli (APC) tumor sup-
pressor gene is known to reduce ODC expression through
suppression of c-myc and activation of its antagonist Mad1
[162]. Participants of the trial, who were homozygous for the
minor A-allele, were 0.48· as likely to have an adenoma
recurrence as individuals homozygous for the major
G-allele. In individuals homozygous for the A-allele and
using aspirin, the relative risk for recurrence was reduced to
0.1· in comparison with individuals homozygous for the
G-allele and not using aspirin [161]. Subsequent experiments
with a human colon cancer-derived cell line indicated that
Mad1 selectively reduces ODC promoter activity possessing
the A-allele but not the G-allele. On the other hand, others
have found that the ODC promoter derived from the minor
A allele more effectively drives the reporter gene than that
from the major G allele [163]. Aspirin was without effect on
ODC but activated SSAT. The apparent synergism between
the polymorphism of the ODC gene and the use of aspirin
was proposed to be a combined result of an inhibition of
polyamine biosynthesis and an activation of their catabol-

ism in human colonic mucosa [161]. Even though these
results are supported by a number of experimental works
suggesting that an inhibition of ODC in combination with
nonsteroidal anti-inflammatory drugs can inhibit colon
carcinogenesis [164,165], it is clearly too premature to assign
an exclusive role for the polyamines in the development of
intestinal tumors.
Spermine synthase
The first documented human polyamine deficiency syn-
drome has been reported very recently. Mutation screens of
families with X-linked mental retardation identified a family
suffering from Snyder–Robinson syndrome that carried a
point mutation in the spermine synthase gene that led to
aberrant splicing, reduced enzyme activity to 5% of normal,
moderately decreased cellular spermine and elevated cellular
spermidine to spermine ratio [166]. The affected males had
mild-to- moderate mental retardation, hypotonia, cerebral
dysfunction, bone abnormalities and facial asymmetry.
These symptoms resemble to some extent, the phenotype
displayed by the Gy mice [148], but are milder. This may be
related to the fact that spermine was totally eliminated in the
tissues of the Gy mice while only partially depleted in cells of
the affected family members [166]. The symptoms were
linked to the ability of spermine to serve as a gateway
molecule for inward rectifier K
+
channels, a function not
fully taken over by spermidine [166].
SSAT
Another X-linked rare syndrome called keratosis follicularis

spinulosa decalvans (KFSD) affecting the skin and eye
may also be related to the metabolism of polyamines. The
syndrome apparently includes a duplication of a region on
the X-chromosome, a region containing SSAT gene [167].
The skin symptoms of the affected individuals resemble
those found in SSAT overexpressing mice. Analyses of
cultured fibroblasts of an affected individual indeed showed
signs of activated polyamine catabolism – three times
increased SSAT activity, enhanced putrescine accumulation
and a decrease in cellular spermidine pool [167].
Concluding remarks
As already demonstrated from studies using pharmacolo-
gical interventions, perturbations of polyamine homeostasis
are remarkably difficult to achieve. Overexpression of ODC
leads to enhanced accumulation of putrescine, yet the
diamine is not converted to the higher polyamines. One way
to force the accumulation of the higher polyamines could be
a hybrid mouse overexpressing ODC and lacking SSAT
activity. Testes appear to be one of the target organs for
ODC overexpression, as high ODC activity ultimately leads
to male infertility. Interestingly, SSAT overexpression leads
to female infertility. Experience with transgenic animals
overexpressing ODC and/or SSAT suggests that putrescine
homeostasis in the skin is critical for hair-cycle. Skin
carcinogenesis can be enhanced through overexpression of
ODC and inhibited by transgenic expression of ODC
antizyme. Polyamine metabolism likewise appears to be
intimately associated with cardiac hypertrophy. Homo-
zygous disruption of ODC or AdoMetDC genesisclearly
incompatible with embryogenesis. This probably applies to

the disruption of the spermidine synthase gene as well.
Spermine deficiency is tolerated relatively well in cultured
cells, but creates a distinct phenotype in whole animals.
Finally, the first examples of human diseases with genetic-
ally altered polyamine metabolism have emerged.
Acknowledgements
The work in the laboratory of the authors was supported by grants
from the Academy of Finland.
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