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Biochemical Society Transactions (2003) Volume 31, part 2
Polyamine dependence of normal cell-cycle
progression
S.M. Oredsson1
Department of Cell and Organism Biology, Lund University, S-223 62 Lund, Sweden
Abstract
The driving force of the cell cycle is the activities of cyclin-dependent kinases (CDKs). Key steps in the
regulation of the cell cycle therefore must impinge upon the activities of the CDKs. CDKs exert their functions
when bound to cyclins that are expressed cyclically during the cell cycle. Polyamine biosynthesis varies
bicyclically during the cell cycle with peaks in enzyme activities at the G1 /S and S/G2 transitions. The
enzyme activities are regulated at transcriptional, translational and post-translational levels. When cells are
seeded in the presence of drugs that interfere with polyamine biosynthesis, cell cycle progression is affected
within one cell cycle after seeding. The cell cycle phase that is most sensitive to polyamine biosynthesis
inhibition is the S phase, while effects on the G1 and G2 /M phases occur at later time points. The elongation
step of DNA replication is negatively affected when polyamine pools are not allowed to increase normally
during cell proliferation. Cyclin A is expressed during the S phase and cyclin A/CDK2 is important for a
normal rate of DNA elongation. Cyclin A expression is lowered in cells treated with polyamine biosynthesis
inhibitors. Thus, polyamines may affect S phase progression by participating in the regulation of cyclin A
expression.
Introduction
In eukaryotic organisms, the size of tissues and organs
depends on a balance between cell proliferation, cell differentiation and cell death. These three processes play an active
role throughout the life of an individual, as they are necessary
for embryogenesis as well as for the functional integrity of
the adult organism. All three processes are strictly regulated
and are highly dependent on a multitude of intracellular and
extracellular factors to progress normally. This paper deals
with cell proliferation and concentrates on the role of the
polyamines putrescine, spermidine and spermine, putting
them into the wider context of various proteins involved
in cell cycle regulation. The basis for cell proliferation is
the orderly progression of the cell through the cell cycle at
the end of which the cell has doubled its content of molecules
vital for life. This ensures the production of two new equalsized healthy daughter cells at the end of a cell cycle.
The cell cycle driving force
Unidirectional progression through the cell cycle is achieved
by an underlying biochemical cycle in which cyclindependent kinases (CDKs) are activated sequentially by
different cyclins [1–4]. The CDKs are constitutively
expressed and are found in excess of their cyclin partners
in the cell [5]. The cyclins on the other hand are expressed
cyclically and the expression of each consecutive cyclin is
Key words: S-adenosylmethionine decarboxylase, cell cycle, cyclin, ornithine decarboxylase,
polyamine.
Abbreviations used: AdoMetDC, S-adenosylmethionine decarboxylase; CDK, cyclin-dependent
kinase; CHO, Chinese hamster ovary; ODC, ornithine decarboxylase; pRB, retinoblastoma protein.
1
e-mail: [email protected]
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dependent on the action of a previously expressed cyclin in
a complex with a CDK. The activity of the cyclin–CDK
complexes is regulated by several mechanisms, including
phosphorylation, dephosphorylation, interaction with CDK
inhibitors and degradation. Although the driving force of the
cell cycle is the phosphorylating activities of cyclin–CDK
complexes, there are intercepting overlying levels of control
called checkpoints [6].
The synthesis of the D-type cyclins begins in response
to mitogenic stimulation in the G1 phase (Figure 1) [7,8].
Cyclin D1 bound to CDK4 mediates the phosphorylation
of a number of proteins of which one is the retinoblastoma
protein (pRB). pRB has a central role in cell cycle progression
from G1 into S phase [9]. In its unphosphorylated form,
pRB sequesters the E2F family of transcription factors that
are required for transcription of genes involved in the G1 /S
transition and S phase progression [10,11]. As pRB is partially
phosphorylated by cyclin D1–CDK4, the transcription factor
E2F-1 is released and stimulates the transcription of cyclin E
in conjunction with the G1 /S transition (Figure 1). Cyclin
E/CDK2 then contributes to the hyperphosphorylation of
pRB. This results in an increased release of E2F-1 from pRB
and stimulation of transcription of genes involved in S phase
progression, e.g. cyclin A (Figure 1). E2F-1 also stimulates
the transcription of genes involved in DNA replication,
like DNA polymerase α, dihydrofolate reductase, thymidine
kinase and thymidylate synthase [10,12].
Cyclin A captures CDK2 from cyclin E when cyclin
E is degraded (Figure 1). Cyclin A co-localizes with sites
of DNA replication in S phase nuclei, suggesting that it
may directly participate in the assembly, activation and/or
regulation of the replication structure [13,14]. In a simian
Polymines and Their Role in Human Disease
Figure 1 Schematic presentation of various factors involved in
cell cycle regulation
Unidirectional progression through the cell cycle is driven by an
underlying biochemical cycle in which CDKs are activated sequentially by
different cyclins. The activities of the polyamine biosynthetic enzymes
ODC and AdoMetDC increase in conjunction with the G1 /S and S/G2
transitions. The enzyme activities are regulated at the transcriptional
(mRNA) and post-translational (half-lives of activities) levels.
S-adenosylmethionine is generated by the action of Sadenosylmethionine decarboxylase (AdoMetDC). ODC and
AdoMetDC are the rate-limiting and highly regulated
enzymes of the biosynthetic pathway. The activities of
ODC and AdoMetDC are subjected to complex regulatory
mechanisms involving transcription, translation and posttranslation [19–21].
Regulation of polyamine biosynthesis
during the cell cycle
virus 40 in vitro replication system, cyclin A was not required
for the formation of the initiation complexes but was required
for efficient DNA elongation [15]. Cyclin A subsequently
switches partner to CDK1 at the end of the S phase (Figure 1).
As cyclin A is degraded at the end of G2 phase, CDK1
captures cyclin B as its partner and entry into mitosis is
induced by increased activity of the cyclin B–CDK1 complex,
which also is known as mitosis-promoting factor [16]. Cyclin
B is rapidly degraded at the end of mitosis (Figure 1).
Polyamine biosynthesis
The polyamines putrescine, spermidine and spermine are
synthesized in well-regulated biochemical pathways [17,18].
The amino acid ornithine is converted to putrescine in
a decarboxylation reaction catalysed by ornithine decarboxylase (ODC). Spermidine is synthesized in a reaction
where a propylamine group derived from decarboxylated Sadenosylmethionine is coupled to putrescine by spermidine
synthase. Spermine is formed from spermidine in a similar
reaction catalysed by spermine synthase. Decarboxylated
The activities of ODC and AdoMetDC have been investigated during the cell cycle in cell populations synchronized
by various synchronization methods [22–25]. The general
conclusion of all these studies is that ODC and AdoMetDC
are activated in a biphasic manner during the cell cycle
with a first activation phase in late G1 close to the G1 /S
transition and a second activation phase in conjunction with
the S/G2 transition (Figure 1). From these results it may be
concluded that ODC and AdoMetDC are not required for
the initial progression through the G1 phase but presumably
for processes during the S phase and thereafter.
It is known that ODC is a target gene for c-Myc [26,27]
and c-myc in turn is a target gene for E2F-1 [10,12]. Thus, the
activation of ODC in conjunction with the G1 /S transition
may be a consequence of the initial phosphorylating activity
of cyclin D1/CDK4 on pRB. Pyronnet et al. [28] found
the second activation peak of ODC at the G2 /M transition,
i.e. somewhat later than other observations [22–25]. They
attributed the second increase in ODC activity to the presence
of a cap-independent internal ribosome entry site in the ODC
mRNA that presumably functions exclusively at the G2 /M
transition.
To investigate the role of transcriptional and posttranslational mechanisms in the regulation of ODC and
AdoMetDC activities during the cell cycle of Chinese hamster
ovary (CHO) cells, we investigated the mRNA levels of
the enzymes and the half-lives of the enzyme activities,
respectively [25,29].
Both ODC and AdoMetDC mRNA levels doubled during
the cell cycle [25]. The doubling of the ODC mRNA content
was found at the end of the S phase in conjunction with
a doubling of the enzyme activity (Figure 1). At the same
time there was a slight increase in the half-life of the enzyme
activity (Figure 1). Thus both transcriptional and posttranslational mechanisms were responsible for the increase
in ODC activity in the late S phase. However, the increase in
ODC activity in conjunction with the G1 /S transition took
place without a change in the ODC mRNA content while
there was an increase in the half-life of the enzyme activity,
implying the involvement of post-translational regulation
(Figure 1).
The doubling of the AdoMetDC mRNA content took
place when there was a doubling of the AdoMetDC activity
at the G1 /S transition (Figure 1). At the same time, there
was also an increase in the half-life of the AdoMetDC
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Biochemical Society Transactions (2003) Volume 31, part 2
activity (Figure 1). Thus, the initial increase in AdoMetDC activity in conjunction with the G1 /S transition was regulated
both transcriptionally and post-translationally. The second
increase in AdoMetDC activity at the S/G2 transition was
presumably regulated at the translational level, as there
was no change in the mRNA content or the half-life of the
enzyme activity (Figure 1). However, this notion has to
be investigated as well as the contribution of translation
in the regulation of enzyme activities of both enzymes during
the entire cell cycle.
Figure 2 Cell cycle progression was affected when mitotic
CHO cells were seeded in the presence of the ODC inhibitor
α-difluoromethylornithine
Cells in mitosis, synchronized by the mitotic shake-off technique, were
seeded in the absence or presence of 5 mM α-difluoromethylornithine.
At various times after seeding, the cells were sampled by trypsination
and fixed in 70% ethanol. The cellular DNA was stained with propidium
iodide and the cell cycle phase distribution of the cell populations
was determined by flow cytometry. 䊐, G1 phase control; 䊊, S phase
control; 䊏, G1 phase with α-difluoromethylornithine; 䊉, S phase with
α-difluoromethylornithine.
Polyamine levels during the cell cycle
Regarding polyamine levels, Heby et al. [30] found clear
biphasic increases in putrescine, spermidine and spermine
levels in CHO cells coinciding with the biphasic ODC
activity during the cell cycle [23]. Other studies have shown
a doubling of the polyamine contents during the cell cycle
without marked fluctuations [25,31–33]. In our study using
CHO cells, the doubling of the putrescine content mainly
took part during late S phase and S/G2 transition, while
the doubling of the spermine content mainly took place
during G1 and S phases [25]. The spermidine level showed
a constant increase during the entire cell cycle. In general,
it seems that the rather large fluctuations in ODC and
AdoMetDC activities results in more subtle changes in
the total cellular polyamine pools. However, there may
be pronounced changes in different intracellular polyamine
pools that presently cannot be distinguished [34,35].
Polyamine dependence of cells
progressing synchronously through
the cell cycle
Cells may be depleted of their polyamines by treatment
with drugs that inhibit ODC or AdoMetDC or stimulate catabolism of polyamines [36,37]. Although there are
numerous publications on the effects of polyamine biosynthesis inhibitors on asynchronously proliferating cells,
results from experiments with inhibitors on synchronously
proliferating cells have not been published. We have
seeded CHO cells synchronized by the mitotic shake-off
technique in the absence or presence of the ODC inhibitor
α-difluoromethylornithine and followed the progression
through the cell cycle using flow cytometry (Figure 2).
At 7 h after seeding, an effect on cell cycle progression
was apparent, as there were fewer cells in the S phase in
α-difluoromethylornithine-treated cultures than in control
cultures. Similar results were found when seeding mitotic
CHO cells in the presence of the spermine analogue N1 ,N11 diethylnorspermine (P.S.H. Berntsson and S.M. Oredsson,
unpublished work). N1 ,N11 -Diethylnorspermine depletes
the polyamine pools mainly by stimulating polyamine
catabolism but also by inhibiting ODC and AdoMetDC
[37]. Thus, unperturbed polyamine biosynthesis is needed
for unperturbed cell cycle progression.
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Because of the overlap in DNA distributions between G1
phase cells and early S phase cells, from these experiments
one can only draw the conclusion that there was a cell cycle
effect but not if the G1 phase was prolonged, if the G1 /S
phase transition was affected or if there was a delay in
S phase progression. Such overlaps in DNA distribution
have a greater impact on the analysis of cell cycle phase
distributions of synchronously proliferating cells than on the
analysis of asynchronously proliferating cells.
Polyamine dependence of cells
progressing asynchronously through the
cell cycle
Numerous studies have been performed with polyamine
biosynthesis inhibitors and polyamine analogues on asynchronously proliferating cells. The result of polyamine
depletion is always growth inhibition, which usually is
reversible when the drug is removed or the cells are
supplemented with polyamines. Polyamine depletion may
also result in apoptosis or senescence [38–40]. When cells
stop proliferating because of polyamine deficiency, different
effects are found on the cell cycle phase distribution. Cells
may accumulate in the G1 phase of the cell cycle [38,41] but
also in S and G2 phases [42–44]. The molecular basis for the
differences in cell cycle accumulation is not clear, but it is most
Polymines and Their Role in Human Disease
probably related to the combination of genetic aberrations in
the cell lines studied. So far there are very few publications
reporting the effects on different cell cycle regulatory
proteins in cells growth-arrested by polyamine depletion.
The N1 ,N11 -diethylnorspermine-induced G1 arrest found in
MALME-3M human melanoma cells was correlated to a
hypophosphorylation of pRB and increased levels of the
tumour suppressor protein p53 and the two CDK inhibitors
p21 and p27 [38]. p53 is involved in the checkpoint control
mechanisms that ascertain the genomic integrity of the cell,
only allowing the production of new healthy daughter cells
[6]. In the human melanoma cell line SK-MEL-28, lacking
functional p53, there was no G1 arrest [38]. In CHO
cells lacking functional p53, polyamine depletion resulted
in an S phase accumulation [45]. However, to draw any
general conclusions about the role of p53 in polyamine
depletion-induced growth arrest, more studies have to be
performed.
It has to be emphasized that effects on specific cell cycle
phases are observed before the onset of growth arrest. Using
a bromodeoxyuridine–DNA flow cytometry method, we
have shown that cell cycle progression is affected within one
cell cycle after seeding cells in the presence of drugs that
deplete the cellular polyamine pools [46–48]. The S phase
was prolonged within the first cell cycle after seeding the
cells, while effects on the G1 /S transition and the G2 +M phase
were found in the following cell cycles. Altogether our results
imply that the delay in S phase progression in polyaminedepleted cells is due to a decreased rate of DNA elongation
rather than to effects on the initiation of DNA replication
[49]. In MCF-7 human breast adenocarcinoma cells treated
with N1 ,N11 -diethylnorspermine, cyclin A expression was
decreased on both the mRNA and protein levels before the
onset of the delay in S phase progression (P.S.H. Berntsson
and S.M. Oredsson, unpublished work). At the same time
point, there were no effects of N1 ,N11 -diethylnorspermine
treatment on the expression of cyclin D1, CDK2, cyclin
E, E2F-1 and the CDK inhibitor p27 or on the expression
or phosphorylation of pRB. Thus, polyamines may be
important in a step between the initial release of E2F-1 due to
the early phosphorylation of pRB by cyclin D1–CDK2 and
the full release of E2F-1 due to hypophosphorylation of pRB,
which results in the transcription of cyclin A. However, this
notion has to be investigated and the effect of polyamine
biosynthesis inhibition on cyclin A expression has to be
studied in other cell lines in order to draw any general
conclusions from this observation in MCF-7 cells.
Concluding remarks
It is clear that normal cell cycle progression requires
unperturbed polyamine metabolism. With the current
knowledge of factors involved in cell cycle regulation and
sensitive methods to study molecular interactions, it should
now be possible to pinpoint steps where polyamines are
required.
I wish to thank Kersti Alm for constructive remarks regarding the
manuscript. My laboratory is supported by grants from the Swedish
Cancer Society, the Royal Physiographical Society in Lund, Sweden,
and the Crafoord Foundation.
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Received 28 November 2002