Journal of Cell and Molecular Biology 1: 57-67, 2002.
Golden Horn University, Printed in Turkey.
57
Polyamines in living organisms
Mustafa Yatin
Harvard Medical School, Massachusetts General Hospital, Department of Radiology, Division of
Nuclear Medicine, Boston MA, 02114, USA
Received 30 June 2002; Accepted 10 July 2002
Abstract
Natural polyamines, putrescine, spermidine and spermine are ubiquitous cell components essential for normal
cellular functions and growth. Chemically these compounds are very simple organic aliphatic cations and fully
protonated under physiological conditions. There is a strong correlation between proliferation rate of the cells and
their polyamine contents. Adjustments of intracellular concentrations of polyamines to physiological requirements
are orchestrated by de novo synthesis, polyamine uptake and catabolic reactions. De novo synthesis can in principle
be substituted by polyamine uptake from extracellular environment. Over accumulation of polyamines is controlled
by release and by a feedback regulation system that involves synthesis of a protein, antizyme that leads to
degradation of ornithine decarboxylase and repression of polyamine uptake. The development of specific
polyamine biosynthesis inhibitors and structural analogues of polyamines have revealed that maintaining
polyamine levels are a prerequisite for animal cell proliferation to occur. The interruption of polyamine
biosynthesis or minimizing the uptake of exogenous polyamines via the polyamine transport system offers
meaningful targets for treatment of certain hyperproliferative diseases, most notably cancer. The polyamines
influence confusingly large number biological processes, yet despite several decades of intensive research work,
their exact functions in living organisms remains obscure. In this review, the current state of scientific knowledge
regarding polyamines, their functions and their metabolism in mammalian cells is presented.
Key words: Polyamine, ODC, AdoMetDC, polyamine analogues, cancer
Canl› organizmalarda poliaminler
Özet
Do¤al poliaminler putresin, spermidin ve spermin hücrenin normal ifllev ve büyümesi için esas olan yayg›n
hücresel bilefliklerdir. Bu bileflikler kimyasal yönden çok basit organik alifatik katyonlard›r ve fizyolojik koflullarda
tamamen protonlanm›fl durumdad›rlar. Hücrenin ço¤alma h›z› ile poliamin içeri¤i aras›nda çok yak›n bir iliflki
vard›r. Poliamin konsantrasyonlar›n›n fizyolojik ihtiyaca göre ayarlanmas› yeni sentez, poliamin al›nmas› ve
katabolik reaksiyonlar aras›ndaki uyumlulu¤a ba¤l›d›r. Yeni sentez prensip olarak hücre d›fl› ortam›ndan poliamin
al›nmas› ile sa¤lan›r. Poliaminlerin fazla birikimi, sal›nmas› ve protein sentezi, ornitin dekarboksilaz y›k›m›na
neden olan antizim ve poliamin al›m›n›n bask›lanmas›n› içeren feedback regulasyon sistemi taraf›ndan kontrol
edilir. Spesifik poliamin biyosentez inhibitörlerinin ve poliaminlerin yap›sal analoglar›n›n geliflimi, hayvan hücre
ço¤almas›n›n meydana gelmesinin poliamin düzeyine ba¤l› oldu¤unu ortaya koymufltur. Poliamin biyosentezinin
kesintiye u¤rat›lmas› veya poliamin tafl›nma sistemi vas›tas› ile d›fl ortamdan poliaminlerin al›nmas›n›n azalt›lmas›,
çok h›zl› hücre ço¤almas›n›n söz konusu oldu¤u belirli hastal›klar›n, en önemlisi kanserin, tedavisinde anlaml›
sonuçlar vermifltir. Poliaminler çok fazla say›daki biyolojik olaylar› etkiler, onlarca y›ld›r yap›lan yo¤un
araflt›rmalara ra¤men, canl› organizmalardaki tam ifllevleri hala aç›k de¤ildir. Bu derlemede poliaminlerle ilgili son
bilimsel veriler, ifllevleri ve memeli hücrelerindeki metabolizmalar› sunulmufltur.
Anahtar sözcükler: Poliamin, ODC, AdoMetDC, poliamin analoglar›, kanser
58
Mustafa Yatin
What are polyamines?
The natural polyamines (PA), putrescine (Put: 1,4diaminobutane), spermidine (Spd: N-(3 aminopropyl)1,4-butadiamine) and spermine (Spm: N,N’-bis
(3-aminopropyl-1,4-butanediamine), are very simple
aliphatic multivalent cations with a primary amine
functional group that are fully protonated under
physiological conditions as essential constituents of
all mammalian cells. One or more of these
compounds are present in every living cell. All
prokaryotic and eukaryotic cells synthesize Put and
Spd. Spm synthesis is, however, is largely confined to
nucleated eukaryotic cells. There are important
interspecies differences in polyamine metabolism,
especially between eukaryotic cells, plants, and some
bacteria and protozoa. In some prokaryotes, only Put
and Spd are synthesized, while in other cases, such as
certain thermophilic bacteria, polyamines with chains
longer than Spm are found. Additionally, in some
parasitic organisms, there exist additional enzymes
that are not present in the host cells and, as such,
provide a target for the design of specific antiparasitic
agents. In general, prokaryotes have higher
concentration of Put than Spd and lack Spm.
Eukaryotes, on the other hand, usually have little Put,
but high concentrations of Spd and Spm (Thomas and
Thomas, 2001; Marton and Pegg, 1995). Historically,
the major pathway for the synthesis of Put and Spd
was first established in microorganisms but was later
found to be similar in animal cells (Tabor and Tabor,
1984).
Polyamine functions
The interesting names putrescine refers to
grow-rotten, the decomposition of organic matter by
bacteria and fungi which results in intense odorous
products, and spermidine and spermine recall the
historical discoveries of these compounds in
putrefying meat and seminal fluid. It has been
reported that, rats immediately bury the dead rat
bodies when in natural decay process bacterial
by-products has perfumed the cadaver with Put; the
same rats also bury wood rat toys that are sprinkled
with Put, indicating olfactory detection of Put is
sufficient enough to trigger this behavior (Coffino,
2001; Pinel et al., 1981). Spm was first PA observed
in human seminal fluids in 1678 by Leeuwenhoek A.
van (1632-1723, extraordinary Dutch scientist who
first ever recorded microscopic observations on living
bacteria on plaques from teeth of two old man who
had never cleaned their teeth in their entire lives)
(http://www.ucmp.berkeley.edu/history/leeuwenhoek.
html). Spm re-discovered several times during the
next 200 years before the chemical structure
[NH2(CH2)3HN(CH2)4NH(CH2)3NH2] was finally
confirmed by its synthesis in 1962. Until recent
decades, relatively few references could be found for
these compounds in biochemical literature, indeed, in
1950, there were only two citations to Spd and four
citations to Spm in chemical abstracts. In contrast to
only six citations in 1950, in the period 1988 to 2001
there were 3500 citations to Spd and 2250 citations to
for Spm in MedLine. During the last few decades
there have been a large number of important studies in
many laboratories (mostly in mammalian systems)
indicating that PA are necessary requirements in
rapidly growing normal and neoplastic cells.
Paradoxically, although more than 300 years have
elapsed since the first written document about the
existence of spermine phosphate crystals in human
semen no one can definitely describe the function of
seminal spermine (Janne et al., 1991).
In a universal classification, the PA belong to a
broader group of biologically active amines together
with the so-called biogenic amines such as serotonin,
histamine, and tryptamine, which are monoamines
having important physiological functions (Tabor and
Tabor, 1984). Although the full repertoire of
biological effects of PA are not fully known; they
influence cellular processes at all stages from gene
transcription to protein synthesis, and are central to
regulation of cell growth and differentiation. There is
a positive correlation between the proliferate activity
of cells and their content and utilization of PA
(Marton and Pegg, 1995; Pegg and McCann, 1994).
Recent studies has revealed that PA levels are
increased in both proliferating cells and extra-cellular
tissue fluids under various inflammatory conditions,
as consequence of excretion during tissue
regeneration and release from damaged or dying cells.
Increased levels of PA biosynthetic enzymes,
polyamine uptake and thus elevated levels of PA have
been demonstrated at highly profilerative neoplastic
Polyamines
cells (cancer), inflammatory sites of infection,
trauma,
neurodegenerative
conditions
and
autoimmune diseases (Igarashi and Kashiawagi,
2002; Zhang et al., 2000).
As it is stated previously in this text, PA are fully
protonated under physiological pH (7.2) and act as
counter-ions for negative charges on RNA and DNA.
Then, the question may be asked as whether all of the
effects of polyamines in cell metabolism can be
explained by simple cationic interactions with
macromolecules. Polyamines regulate nucleic acid
conformation in vitro and may have similar role in
vivo. Why, if so, would the cells need to produce
energetically very expensive polyamines through
highly sophisticated pathways, when two Ca2+ or two
Mg2+ would have the same number of positive
charges? Possibly, the major advantage of the
polyamine pathway is that cell can control both the
synthetic production and degradation of the
polyamines as needed independently of the
availability from extracellular environment, a
situation quite different from which is relevant to
other cations having only extracellular origins. Unlike
the point charges of Mg or Ca, the positive charge in
PA distributed along the flexible carbon chain which
may enable the PA uniquely to bridge critical
distances. This unique molecular topography and
distribution of positive charge in PA allow specific
counter-ion interactions that neutralize the negative
charges of phosphates in DNA helices (Thomas and
Thomas, 2001; Vijayanathan et al., 2001; Feurerestein
et al., 1991; Wang et al., 2001). PA’s high positive
charge also prevents them from crossing biological
membranes by simple diffusion (Bergeron et al.,
1995). Further progress in polyamine nucleic acid
interaction in the regulation of transcription or
synthesis depends on multidisciplinary studies
involving cell biologists, biochemists, and physical
and theoretical chemists.
Polyamine metabolism
Cellular polyamines accumulate via coordinated
interactions between de novo synthesis and
transmembrane uptake (Janne et al., 1991; Marton
and Pegg, 1995; Seidenfeld, 1985; Quemener et al.,
1994). As shown in Figure 1, the complete
59
Figure 1: Polyamine biosynthesis. The natural PA in
mammalian and plant cells are Put, Spd and Spm. Some
microorganisms, including trypanasomes, contain only
trace of Spm or may lack it completely. The four key
enzymes making up the PA pathway in mammalian cells are
ornithine decarboxylase (ODC) that forms Put from
L-ornithine;
s-adenosylmethionine
decarboxylase
(AdoMetDC) that forms decarboxylated (dcAdoMet),
which act as an aminopropyl donor; spermidine synthase
that transfers the aminopropyl group from dcAdoMet to
putrescine; and spermine synthase that transfers the
aminopropyl group from dcAdoMet to spermidine. In some
plants and bacterial arginine decarboxylase (b-ADC)
initiates an alternative two-step pathway to putrescine. The
retroconversion of Spm back to Put can be accomplished
by the sequential action of two enzymes, polyamine
oxidase (PAO) and spermidine-spermine acetyltransferase
(SSAT). SSAT-PAO pathway arranges PA pool
composition and becomes particularly important in
preventing PA levels from getting too high after excess
synthesis or uptake as highyl inducible SSAT leads to a
rapid conversion of PA to N1-acetylspermine (N-acetylSpm)
and N1-acetylspermidine (N-acetylSpd), which are readily
excreted from cells.
metabolisms of the polyamines involve some seven
enzyme reactions, each of which precisely regulated
in order to maintain optimum intracellular
concentrations in accordance with cellular needs. In
addition, there are also polyamine transport processes
both into (influx) and out (efflux) of the cell (Marton
and Pegg, 1995; Pegg and McCann, 1994). Again,
these polyamine accumulation activities are highly
controlled with a strong link to the up and down
60
Mustafa Yatin
regulation of cell growth depending on need. The
polyamine requirement of a given cell may be
covered by de novo synthesis or by uptake from its
environment. Under physiologic conditions, the
relative importance of uptake and de novo synthesis is
a cell-typic character (Marton and Pegg, 1995; Seiler
and Dezeure, 1990; Seiler et al., 1990). The capacity
of cells for de novo synthesis and uptake is an
expression of their ability to adapt to environmental
changes.
The use of polyamine levels in body fluids as
diagnostic markers or as indices of novel therapeutic
effects has also been subject to extensive study but the
results have, with a few exceptions, been
disappointing. The extreme complexity of blood
compartment carrying free polyamines in the plasma
caused significant problems in the clinical
interpretation of circulating PA levels. By the end of
1970s the unclear potential role of PA as markers for
cancer led to an almost total disinterest in their
diagnostic use (Moulinoux et al., 1996). However,
studies of polyamine metabolism in a number of
pathogenic parasites (Seiler and Atanassov, 1994),
inflammatory, infectious conditions (Yatin and
Fischman, 2002) and under oxidative stress (Gilad
and Gilad, 1999), have led to identification a number
therapeutic targets and the development of novel
chemotherapeutic agents (Pegg and McCann, 1994;
Wallace and Morgan, 1990; Hebby, 1981; Seiler and
Atanassov, 1994). Polyamines are also modulators of
synaptic functions and play important roles in central
nervous system (CNS) (Gilad and Gilad, 1999; Yatin
et al., 2001; Williams, 1997). Some of the
non-peptide venoms of spiders and wasps are natural
polyamine analogues that are selective inhibitors of
the glutamate receptors of the CNS (Nihei et al.,
2001; Palma et al., 1998; Albensi et al., 2000).
Many plant compounds contain polyamine
residues, including several families of alkaloids (Hoet
and Nemery, 2000; Thomas and Thomas, 2001).
Resveratrol, a natural polyphenolic phytoalexine,
present abundantly in red wine, has been reported to
be an anti-proliferative agent on human cancer cells,
also caused significant decreases on ODC activity,
indicating that PA might represent one of several
beneficiary effects of moderate consumption of red
wine (Nigdigar et al., 1998; Scheneider et al., 2000).
Spm, exceptionally high in skin (human skin
concentration - epidermis is 850 mg/g tissue
compared to muscle - skeletal, 30 mg/g tissue), has
been identified as a potent antioxidant and
anti-inflammatory agent against UVB irradiation and
oxidative stress and suggested to be an important
anti-inflammatory antioxidant of epidermis.
Currently, the use of Spm as a dermatologic
antioxidant is under patent protection (Lovaas, 1995).
Biosynthesis and regulation of cellular polyamines
The biosynthesis pathway of polyamine accumulation
in the cells is kinetically rate-limited by ornithine
decarboxylase (ODC; EC 4.1.1.17), a pyridoxal
phosphate dependent enzyme, which synthesize Put
by decarboxylation of L-ornithine. The remarkable
elevation in de novo polyamine biosynthesis rate that
takes place in rapidly growing cells has led to much
effort to develop therapeutically useful antineoplastic
agents that would interfere with, or regulate, these
processes in hyperproliferative diseases (Seiler and
Atanassov, 1998). Investigation of several specific
inhibitors of ODC (i.e. difluoromethylornithine,
DFMO, a suicidal irreversible specific inhibitor of
ODC) as an experimental antineoplastic strategy
causes to reduction in total amount of cellular
polyamines (except spermine) with only cytostatic
effects. The lack of cytotoxicity may be due to
polyamine interconversion from a spermine reservoir
in the cells and/or polyamine repletion by
transmembrane uptake from extracellular sources
(Pegg and McCann, 1994; Hebby, 1981; Seiler and
Atanassov, 1998).
Many microorganisms and higher plants are able
to biosynthesize Put from agmatine produced by
decarboxylation of arginine, however all mammalian
cells and many lower eukaryotes lack arginine
decarboxylase (ADC), leaving the only route to
produce Put to utilize the ODC. Ornithine is available
for this enzymatic reaction from the circulating
plasma and can also be formed by the action of
arginase. Arginase is much more widely distributed
than any other enzymes of the urea cycle in living
organisms and present in extrahepatic tissues, and
ensures the availability of ornithine in PA production
line. In a broad sense, arginase can, therefore be
assumed of as an initial step in the PA biosynthesis
pathway.
Ornithine decarboxylase was discovered in 1968
simultaneously and independently in two laboratories
in the United States and one in Finland (Janne et al.,
Polyamines
61
Figure 2: Cellular PA pool is regulated by three influences: production, transport and catabolism. Antizyme has a negative
feedback control on production and transport. Antizyme is a key factor in cellular PA homoestasis and its production depends
on PA level. When cellular PA levels rise, +1 frame-shifting in AZ ribosomal mRNA occurs causing read-through of the
internal stop codon to produce the full length antizyme protein. ODC is active and stable enzyme, if only, it forms a
homodimer in trans form across the face of the active dimer. Higher affinity of AZ towards ODC, generates an AZ-ODC
heterodimer exposing the C-terminus of ODC, which is then immediately recognized and degraded by the 26S proteosome.
In contrast with all other cellular proteins degraded by this proteolytic pathway, the degradation of ODC is not triggered by
ubiquitination. AZ also reversibly binds to some functional component of the cytoplasmic membrane PA uptake transporter and
thereby preventing utilization of extracellular sources of PAs. AZ by itself can be bound by an AZ inhibitor, which is an
ODC-like protein that does not posses ODC activity but which does bind AZ more strongly than the AZ-ODC complex, and so
consequently sequesters the AZ protein. In this negative feedback loop; cellular PA rise to excessive amounts; PA induce more
AZ; AZ inhibits and diminishes ODC and PA uptake; PA accumulation in cells decline.
1991, Russel and Synder, 1968; Pegg and Williams,
1968). Ornithine decarboxylase is a unique enzyme in
many respects. First, it is one of the enzymes having
extremely short life with a very rapid turnover rate
(t1/2 is from 10 to 20 minutes) and it is present in very
small amounts in normal growing cells. Its activity
can be increased many folds within a few hours of
exposure to trophic stimuli (Janne et al., 1991). Such
stimuli include hormones, various drugs, tissue
regeneration and growth factors commonly found in
serum. Even after such stimulation, ODC remains
only a very small fraction of the total cellular protein
ranging from 0.01% of the cytosolic protein in
androgen - stimulated mouse kidneys to 0.00012 % in
thioacetamide stimulated rat liver. It turned out that
by any definition this enzyme is a low abundance
protein representing only about 3 ppm (part per
million) of the soluble proteins of a mammalian cell.
Second, treatment of cells with exogenously added
polyamines cause negative feedback and result in a
rapid and profound fall in enzymatic activity of ODC.
Cannelakis (1989) found that the loss of ODC activity
coincided with the appearance of another enzyme
activity that was inhibitor of ODC. This activity was
called anti-enzyme for ODC or more briefly antizyme
(Coffino, 2001; Heller et al., 1976). The low
abundance of both proteins presented tremendous
challenge in studying their isolation and biochemical
nature, physiological behavior and chemical
mechanism of their interaction. Shin-Ichi Hayashi,
addressed the problem by purifying ODC to
homogeneity, a very difficult task at that time, to
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Mustafa Yatin
show that pure ODC enzyme form stoichiometrically
1:1 complex that is enzymatically inactive, but then
can be reversed to dissociate to regenerate ODC
activity (Figure 2) (Hayashi et al., 1996). The cell
culture studies followed this study to come with
finding that the antizyme:ODC ratio was strongly
correlated with the degradation rate of ODC (Coffino,
2001).
Following Hayashi’s achievement of cloning of
the antizyme cDNA and gene, it was promptly
showed that transfection of cells to overexpression of
antizyme significantly caused ODC activity and ODC
protein to fall. The protease that is responsible from
this destructive process was shown to be proteosome.
This was later established definitely that antizyme
promotes the destruction of ODC by the 26 S
proteasome in an in vitro system (by using only
purified components) (Murakami et al., 1992). The
complete mechanism of antizyme action were then
mechanistically confirmed with +1 translational
frameshifting in antizyme mRNA by polyamines
(Coffino, 2001).
The next regulatory enzyme in the polyamine
metabolic pathway is s-adenosyl-methionine
decarboxylase (AdoMetDC; EC 4.1.1.50) that
provides aminopropyl groups for the synthesis of
higher polyamines, spermidine and spermine, from
first polyamine putrescine. The aminopropyl moiety
is derived from methionine, which is first converted
into s-adenosylmethionine (AdoMet) and is then
AdoMetDC produces decarboxylated adenosylmethione
(dcAdoMet). The half life of AdoMetDC is relatively
longer than that of ODC but is still only about 30 to
60 min. The substrate AdoMet is an important methyl
donor (for DNA methylation via methyl transferase)
in eukaryatic cells and AdoMetDC act as a regulatory
control point in AdoMet netabolism. dcAdoMet is
essentially inactive as a methyl donor, so once
AdoMetDC converts AdoMet to dcAdoMet, it is
away from all other metobolic paths and can be
utilized only in PA biosynthetic pathway (Stanley and
Shantz, 1994).
Since excessive PA accumulation in cells is
harmful for normal cell function, both PA
biosynthesis and PA transport are expected to be
tightly feedback-regulated by a common mechanism
(Morris, 1991). In cell culture studies,
polyamine-deprived cells rapidly internalize
exogenously administered PA until intracelleular PA
levels are replenished fully, generally within 1-3
hours of PA addition, and then uptake is abruptly
terminated (He et al., 1994). The red flag signal for
this feedback response requires active protein
synthsesis and it is strongly correlated to presence of
excess spermidine and spermine and several PA
analogues. Negative regulation of PA transport by
antizyme was demonsrated in ODC overproducing
cells and DFMO treated hepatoma cells transfected
with antizyme cDNA (He et al., 1994). Although
substantial progress has been made in understanding
feedback regulation of ODC, much less is known
about antizyme mediated feedback repression of PA
transport (Mitchell et al., 1994).
Polyamine transport
Although a tightly regulated biosynthetic pathway
largely produces intracellular polyamines, a large
number of cell types from different species (both
prokaryotic and eukaryotic) have been shown to
posses polyamine uptake system, which, under
needed conditions, can substitute for de novo
synthesis. In evolutionary terms, PA transport serves
as an adaptational response of cells to changes in PA
requirement. Cellular uptake mechanisms usually
salvage polyamines from diet and intestinal
microorganisms (Figure 3). In mammalian organism,
PA are taken up from gastrointestinal tract, and
released with the urine (Quemener et al., 1994).
Transmembrane transport of extracellular
polyamines can be enhanced by growth factors and
hormones, as well as by inhibition of intracellular
biosynthesis (i.e. ODC inhibition by DFMO)
(Seidenfeld, 1985; Lessard et al., 1995; Byers and
Pegg, 1989). The mechanisms by which polyamine
uptake is induced are not clear, although it is known
that uptake of polyamines is generally low in
quiescent cells, in contrast to cells rapidly
proliferating or in cells that have been induced to
differentiate and PA utilization is greatly enhanced.
As a general rule, increases in cellular growth is not
only accompanied by enhanced rates of intracellular
de novo synthesis, but also by enhanced rates of
uptakes of exogenous PA (Seiler and Dezeure, 1990;
Seiler et al., 1996).
PA are incorporated by a process that is energy
requiring, temperature-dependent, capable of
accumulating against a substantial concentration
gradient (not via a simple diffusion), saturable and
Polyamines
Figure 3: Several sources of PAs are available for rapidly
growing cells in vivo. The endogenous sources arise from a
highly regulated intracellular metabolism and the exogenous
sources arise principally from the gastrontestinal (GI) tract.
Intestinal and colonic mucosa absorb PA released from food
and from microfloral biosynthesis as well. PA
originating from different source are finally transported by
circulating blood, particullarly in red blood cells (RBC) in
normal conditions or PA can be accumulated at situ under
inflammatory conditions via migration of activated white
blood cells (WBC). When needed, a membrane transport
system allows the host cells to uptake PA from
extracellular compartments.
carrier-mediated (Seiler and Dezeure, 1990; Seiler et
al., 1996; Aziz et al., 1994; Toursarkissian et al;
1994). Although, not studied in most cell types, a
human gene for polyamine transport has been
expressed in polyamine deficient Chinese-hamster
ovary (CHO) mutant cell line (Byers and Pegg, 1989;
Hyvonen et al., 1994). Many cells posses single
transmembrane carrier capable transporting Put, Spd
and Spm. However, in one quarter of all cell types
examined (reports from competition studies) suggests
that more than one carrier (or in general terms, more
than one transporter for polyamine uptake into the
cells; one for Put only and one for Put, Spd and Spm)
is present (Aziz et al., 1994; Toursarkissian et al;
1994; Minchin et al., 1991). Different cells posses
both saturable, and non-saturable uptake systems.
Polyamine uptake by saturable systems is temperature
dependent and carrier-mediated. The presence of
unsaturable components in the polyamine transport
system in some cell types has been reported, but the
evidence is based on the results of statistical non-linear
63
regression analysis and computer-fitting of the data to
the appropriate equations (Minchin et al., 1991).
In general, the affinity of the transporter carrier
increases from Put, Spd and Spm. In all cell types
investigated PA transport is independent and distinct
from amino acid transport systems and in some cells
it appears to be sodium-dependent (Palacin et al.,
1998). Although uptake is observed, in the absence of
Na+, the exogenously increase of sodium in the
medium to physiological concentrations (120 mM)
usually increase the transport rate by 60%.
Modulation of the Na-dependent portion by
ionophores (gramicidin or monensin) inhibits
sodium-dependent portion of PA uptake (Khan et al.,
1992). In addition, natural polyamines, Put, Spd,
Spm, a large number of polyamine analogues can also
be taken up by this system. This lack of specificity of
polyamine uptake systems in the cells have been
made possible exploitation of polyamine-like drugs
for use in cancer chemotheraphy or in other
chemotherapeutic approaches to various diseases
(Kramer et al., 1993).
The structural tolerances of the PA transport
systems allowed the selection of cells resistant to the
cytotoxic action of MGBG, which after limited
mutagenesis had lost their ability to take up PA from
the environment. Uptake-deficient mutants have
widely been used to characterize the uptake systems
or non-specific uptakes used by analogs with
structural relationships to PA. One example to these
uptake-deficient mutant CHO cells is CHO-MGBG
cells. Studies with CHO-MGBG cells also revealed
that PA uptake and export mediated by different
transport systems (Put release was observed in
uptake-deficient CHO-MGBG cells) (Hyvonen et al.,
1994). This may not necessarily imply that the
transporter proteins for uptake and release are
different. Uptake deficient mutants may not simply
lack an active transporter protein, but another
important constituent activating uptake part only
(Seiler et al., 1996).
ODC and proto-oncogenes
Various carcinogens, mitogen stimuli and tumor
promoters may cause transient increases in ODC
activity, while rapidly proliferating tissues, including
tumors, activated macrophages and the cells of gut
mucosa have constantly elevated ODC activities and
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Mustafa Yatin
enhanced rates of PA uptakes that leads to elevated
levels of PA. ODC also exhibits properties of
oncogens, like c-myc, c-fos, c-jun, and expression of
oncogens like src, neu, and Ha-ras result in
significant increases of ODC activity. Similarly,
inhibition of polyamine biosynthetic enzymes by
specific inhibitors and depletion of PA levels is
strongly associated with decreased transcription of
the c-myc, c-fos and ODC gene. Inserting a partial
cDNA coding for ODC under the control of a strong
viral promoter and by transfecting the plasmid to cells
caused to stable ODC over-expression (Thomas and
Thomas, 2001; Janne et al., 1991; Marton and Pegg,
1995). These transfected ODC over-expressed cells
showed malignant transformation and upon
inoculation to nude mice, they produced extensively
vascularized, aggressive tumors (Thomas and
Thomas, 2001). Thus, these evidences from in vitro
and in vivo studies suggested that oncogenes may
enhance the transcription of the ODC gene and certain
oncogens induce ODC and enhance the formation of
PA, and vice versa, PA induce expression of
oncogens.
Polyamine synthesis inhibitors
Blockade of ODC enzyme activity by DFMO causes
a time dependent decrease of the cellular Put level,
followed by a decrease of Spd, in contrast, Spm
concentrations are usually not much effected and even
may increase. The decreases in Put concentration is
obvious due to impairment of its producing enzyme
ODC. The depletion of Spd has mainly three reasons:
first, decreased formation due the limited availability
of Put as substrate of Spd synthase. Second, dilution
of PA pool due to cell division and decrease in the
amount per cell. Third, enhanced formation of Spm
due to elevation in AdoMetDC activity. The reason
for the induction of AdoMetDC in DFMO treated
cells (thus Spd depleted) is the unavailability of Put as
substrate. This consequently causes to excessive
availability of dcAdoMet and leads to accumulation
of Spm in the cells.
AdoMetDC is effectively inhibited by
methylglyoxal bis (guanylhydrazone) (MGBG) and
this drug can be also be to inhibit PA synthesis (to
decline Spd and Spm levels) in vivo. However,
MGBG is not specific to AdoMetDC, it also inhibits
diamine oxidase (DAO) and has chemical structre
with considerable resemblance to PA and is taken up
by the same transport system as PA. Therefore, it is
difficult to prove the antiproliferative effects of
MGBG are due to PA depletion as its effects can be
reversed by exogenous addition of Spd could simply
be due to displacement of MGBG from intracellular
sites by structuraly similar Spd or competitive
interference with drug transport. Additionaly,
therapeutic combination of MGBG with DFMO
causes to abnormal accumulation in the cells and
non-specific cytoxicities. In recent years, industrial
research programs (Ciba-Geigy) developed structural
derivatives of MGBG to minimize non-specific
effects. The bicyclic analog of MGBG,
4-(aminoiminomethyl)-2,3-dhydro-1 Hinden-1-onediaminomethylenehydrazone (CGP-48664) fulfilled
this criterion, eliminating the severe toxicities
associated with parent compound. Mutant cell lines
lacking PA transport are resistant to MGBG, but they
remain sensitive to CGP-48644 (Thomas and Thomas,
2001). Thus, CGP-48644, not like MGBG, does not
share the same uptake mechanism with PA, allowing
combination with DFMO to deplete all three PA levels,
has a much broader therapeutic window than MGBG.
The back conversion of Spm to Spd and to Put is
mediated by dual actions of N1-acetylation (SSAT)
followed by oxidative removal acetamidopropanal by
polyamine oxidase (PAO) to maintain the proper
balance of PA pools to ensure cell growth (Figure 1).
Although no inhibitor is developed to specifically
inactivate SSAT, a series of compounds have been
designed to inhibit PAO very potently and efficiently
(Bolkenius et al., 1985). The most widely used of
these inhibitors is the polyamine analogue, N1-N2-bis
(2,3-butadienyl)-1,4-butanediamine (MDL-72527).
Cells treated with MDL-72527 alone are not growth
inhibited, indicating that the back conversion pathway
is not a critical step for cell growth under normal
conditions. However, when MDL-72527 was
administered in combination with other PA enzyme
inhibitors (i.e. DFMO) or PA analogues, a much greater
depletion of PA pools were achieved and caused
significant growth inhibition than either agent given
alone (Claveria et al., 1987; Prakash et al., 1990).
Polyamine analogues
Porter and Bergeron(1988) were the first to suggest
the use of polyamine analogues as a new approach in
Polyamines
chemotherapy and lead to a very large number of
structural analogues and homologs with the general
formula: R1-NH-(CH2)a-NH-(CH2)b-NH-(CH2)-NHR2, where R1 and R2 are alkyl residues, and a and b is
any integer number. In general, polyamine analogues
competitively use the same transport system with PA
to incorporate cells, interferes with PA metabolism,
lacks the necessary physiological functions of PA in
normal cellular functions, but suppress ODC and
AdoMetDC activities and induce SSAT activities,
leads to rapid depletion of PA levels (Bergeron et al.,
1997).
Starting in mid 1980s efforts to synthesize and
identify specific PA inhibitors have began in several
academic and industrial laboratories. The structural
characteristics of these PA transport inhibitors (i.e.
compounds which deter or compete with natural PA
for transport) have been analyzed by QSAR and by
COMFA (comparative molecular field analysis) as
well as simple charge to chain length correlation
produced a preliminary theoretical predictive model
to design new analogues (Xia et al., 1997; Li et al.,
1997; Burns et al., 2001).
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