Polyamines: Specific Metabolic Regulators or Multifunctional Polycations?
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Received 1 July 1998
Polyamine analogue-mediated cell cycle responses in human melanoma cells involves
the p53, p2 I, Rb regulatory pathway
D. L. Krarner, S. Vujcic, P. Diegelman, C. White, J. D. Black and C. W. Porter
Roswell Park Cancer Institute, Elm and Carlton Street, Buffalo, N Y 14263, U.S.A.
T h e intracellular abundance and broad biological
distribution of polyamines among various plant
and animal cell types has generated long-standing interest in understanding their cellular function(s). Although the critical role of these
interesting molecules in initiating and maintaining cell growth is now well documented, the precise growth-related molecular events which they
are presumed to mediate remain to be determined. T h e importance of such information is
further indicated by the recognition that polyamines have been shown to represent a meaningful target for treating or preventing diseases
involving uncontrolled cell growth [ 1-31. Novel
polyamine analogues designed to dysregulate
polyamine metabolic responses as a means to
achieve polyamine pool depletion and cell growth
inhibition have been developed [2]. In response
to such analogues, cells typically down-regulate
Abbreviations used: DENSPM, N',N"-diethylnorspermine [also known as DE-333 or BENSPM as in
N',N"-bis (ethy1)norsperminel; p16, ~16'"'~; p21,
p21Wafl/C~pl;
P27,
~27~'~';
Rb,
retinoblastoma protein; SSAT, spermidine/spermine
N1-acetyltransferase.
polyamine biosynthetic enzyme and transport
activities, while at the same time potently upregulating the polyamine catabolic enzyme, spermidinelspermine N'-acetyltransferase
(SSAT)
[4].T h e net result is the replacement of natural
polyamines with an analogue which by design, is
incompatible with growth-related functions. O n
the basis of promising activity in model tumour
systems [5,6], certain spermine analogues, such
as N',"'-diethylnorspermine
(DENSPM) , have
progressed to clinical trials.
Because polyamine analogues, such as
DENSPM, are capable of substituting for natural
polyamines in at least some regulatory functions
[3], they provide a unique opportunity to explore
polyamine function in cell growth while at the
same time gaining mechanistic information
needed for their rational clinical deployment and
for the design of new and more effective analogues. Towards these aims, we have focused on
determining the impact of DENSPM and related
analogues on cell cycle progression, giving particular attention to newly delineated pathways
which regulate that process. Despite having
co-existed for several years, analogues have not
I998
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yet been examined in the context of these complex regulatory pathways. As shown in Figure 1,
altered expression of proteins involved in regulating these pathways is capable of contributing
significantly to the transformed phenotype [7].
More specifically, overexpression of oncogenes
involved in signal transduction and cell cycle
regulation and inactivation of growth suppressor
genes drives transformed cells to ignore normal
cellular response options such as differentiation,
senescence and quiescence in favour of uncontrolled growth. By examining how polyamine ana-
logues interfere with cell growth in tumour types
of differing genotype we hope to localize polyamine-related decision points and thus further
our understanding of their role in cell growth.
These studies were initiated by our recent observation [8] that MALME-3M human melanoma
cells treated with certain polyamine analogues
undergo GI and G2/M arrests which were eventually followed by apoptosis. The distinct nature
of the GI arrest provided the opportunity to
examine the underlying regulatory responses
which typify a cell cycle checkpoint for such a
Figure I
Role of oncogenes and cell cycle proteins in determining growth-related cell
behaviour
Normal cells (upper panel) have a balanced cell cycle regulatory system and may proceed to
differentiation, senescence, quiescence or controlled cell growth, whereas transformed cells
(lower panel) have an imbalance of cell cycle regulatory mechanisms and are driven towards
uncontrolled cell growth. Adapted with permission from DelSal et al. [7], Cell Cycle and
Cancer: Critical Events at the GI Restriction Point, Copyright 1996, published by Begell
House, Inc.
I'
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Polyamines: Specific Metabolic Regulators or Multifunctional Polycations?
response. Here, we summarize the findings of
studies reported elsewhere in greater detail [8,9].
DENSPM-induced GI arrest
A detailed characterization of time-dependent
DENSPM effects (at 10 pM) on asynchronously
growing MALME-3M cells [8,9], revealed a significant increase in the G I phase population and
a concomitant decrease in the S phase cell population which was apparent first between 16 and
20 h. By 48 h, cell cycle arrest had proceeded to
completion with GI phase cells accounting for
approx. 90% of the total cell population and S
phase cells accounting for <2%; GJM cells
represented approx. 9%. Having established the
kinetics of growth arrest as detected by flow cytometry, we next sought to determine which regulatory pathway was involved, focusing on the p53
network [10,11] and initially, on the Rb protein.
It is well recognized that the phosphorylation
status of the retinoblastoma (Rb) protein controls its ability to release the transcription factor
E2F which, in turn, activates various S phasespecific genes [10,11]. Consistent with the cell
cycle data, Rb was found by Western blot analysis
to be predominantly hyperphosphorylated (ppRb)
until 16 h at which time a shift to the hypophosphorylated state (pRb) became apparent. By 24 h,
the pRb was the predominant species and by
48 h it was the only visible species. These data
suggested an inability of the cyclin/cyclindependent kinase complex to phosphorylate Rb,
an event frequently blocked by the cyclindependent kinase inhibitor, p21Waf”Clpl
(P21) [121.
Western blot analysis revealed a time-dependent
increase in p21 which began at 16 h and reached
a 5-fold maximum at 30 h. Another cyclindependent kinase inhibitor, ~ 2 7 ~[ 121
’ ~ was
’ relatively unaffected and p16 was not detected. One
upstream regulator of p21 is known to be p53
[9,10], which was found to increase earlier than
p21 (i.e. at 12 h) reaching a 10-fold maximum at
30 h. Thus, the GI arrest associated with
DENSPM treatment appeared on the basis of
temporal relationships to involve the p53, p21,
Rb pathway. T o further substantiate the role of
this pathway, we examined mRNA levels by
Northern blot analysis. p53 mRNA was
unaffected during DENSPM treatment which is
consistent with post-transcriptional activation as
it is known to occur via the DNA damage pathway [13]. In contrast, p21 mRNA increased
substantially in a manner consistent with trans-
activation of the gene by induced p53 which
serves as a transcription factor [10,11].
We next examined DENSPM effects on synchronized cell populations in order to exclude
the possibility that the increases in protein levels
may be secondary to accumulation of cells in GI
as opposed to being responsible for the latter.
Following 120 h of serum starvation, MALME3M cells accumulated in G I As shown in Figure
2. T h e cells were then released in complete
medium in the presence or absence of 10pM
DENSPM. T h e untreated control cells moved
through S phase at 24 h and re-established a
normal profile by 48 h. T h e treated cells progressed through S following a slight delay and by
48 h eventually partitioned between GI (80%)
and G2/M (20%) with no detectable cells in S
phase. As a similar population profile was
obtained at 6 0 h and because there was no
increase in cell number, the findings were indicative of a G I and G2/M cell cycle arrest. T h e
changes in Rb, p21 and p53 detected by Western
Figure 2
Cell cycle analysis and Western blot analysis of p53,
p21 and Rb
MALME-3M human melanoma cells were synchronized by serum
deprivation for I20 h and then released in serum-containing
media in the presence or absence of I0 pM DENSPM.
1
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Biochemical Society Transactions
612
blot analysis were consistent with this interpretation (Figure 2). Serum addition to the control
cells shifted Rb from the hypophosphorylated
state to the hyperssphorylated state, which
remained constant for the next 60 h. Levels of
p53 and p21 in control cells were unchanged.
Cells released in the presence of DENSPM contained sufficient ppRb at 24 h to account for an
initial transit into S phase. However, following
release, both p53 and p21 were induced at 24 h
and reached a maximum at 48 h, at which time
pRb again became the predominant species.
Thus, although DENSPM allows cells to initially
transit S phase, they arrest within one cell doubling with a greater proportion remaining in G2/M
(approx. 20%) than was observed for treated
asynchronous cells (approx. 10%). Thus in both
asynchronous and synchronous MALME-3M
cells, DENSPM treatment caused a specific
accumulation of GI and GJM phase cells via
activation of the p53, p21, Rb pathway.
DENSPM-induced apoptosis
MALME-3M human melanoma cells are known
to contain wild-type p53 protein [14]. T o investigate further the role of p53 in DENSPMmediated G I arrest, studies were conducted in a
humafi melanoma cell line, SK-MEL-28, known
to contain a p53 bearing a single amino acid
Figure 3
Protective effect of p53-mediated GI arrest following a toxic cellular insult,
such as DENSPM exposure
Cells containing wild-type p53 (upper panel) undergo a p53-mediated GI arrest which offers
an opportunity to recover from insults. In contrast, cells containing mutated p53 (lower
panel) fail to arrest and proceed immediately to apoptosis.
Treated w t p53 TUmor
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Polyamines: Specific Metabolic Regulators or Multifunctional Polycations?
mutation in the DNA binding region of the
molecule [14]. In response to DENSPM treatment, these cells became rapidly growth
inhibited (i.e. <24 h) and then proceeded to
undergo near-total apoptosis by 48 h; this was
assessed by morphological evaluation of nuclei
and by NMR spectral changes in the membrane
lipids of intact treated cells [15,16]. This rapid
apoptotic response was not preceded by an obvious GI arrest as indicated by flow cytometric
analysis. Furthermore, there was no detectable
induction of p53, p21, or other cyclin-dependent
kinase inhibitors, including p27 or p16 [12].
These findings are in sharp contrast to those
obtained with MALME-3M, which gradually
developed a G1 arrest before undergoing delayed
and limited apoptosis (i.e. after 72 h). Based on
these findings, we conclude that DENSPM treatment probably induces a p53-dependent G1
arrest in MALME-3M cells, and p-53independent apoptotic pathway in the SK-MEL28 cells, which has not yet been identified.
DENSPM induction of SSAT
In both MALME-3M and SK-MEL-28 cells,
DENSPM treatment causes a rapid depletion of
polyamine pools and a massive induction (i.e.
>700-fold) of the catabolic enzyme SSAT. It was
of interest to determine whether either or both
of these events are causally related to the cell
cycle and apoptotic effects of the analogue;
initially we focused on the SK-MEL-28 cells,
owing to the unequivocal nature of the apoptotic
response. For this purpose, DENSPM and a
series of three structurally related aliphatic analogues were used [8,17]. T h e analogues are
known to accumulate to comparable intracellular
levels, similarly deplete polyamine pools but differentially induce SSAT [8,10]. Thus if polyamine depletion or some unidentified analogue
effect is critical, all analogues should affect apoptosis similarly whereas if SSAT is critical, the
analogues should affect apoptosis differently. In
agreement with the latter prediction, a striking
correlation was observed between analogue
induction of SSAT and the ability to induce apoptosis in SK-MEL-28 cells. Thus, we conclude
that SSAT induction is a critical underlying
event in the cell cycle events produced by
DENSPM. Possible downstream consequences of
SSAT overproduction include hydrogen peroxide
excess [18], depletion of acetylCoA pools, altered
acetylation of histones [ 181, altered acetylation of
p53 [ 19,201, and/or other unforeseen events.
Conclusions
613
T h e findings summarized here indicate that the
polyamine analogue, DENSPM, potently activates the p53/p21/Rb cell cycle regulatory pathway. Cells containing wild-type p53, such as
MALME-3M, respond by undergoing a G1 and
G2/M cell cycle arrest which could theoretically
allow time for recovery and protection from cell
death. In contrast, cells containing a mutated p53
such as SK-MEL-28 forego this protective arrest
and proceed immediately to massive apoptosis.
This second finding could be clinically relevant
because approx. 50% of human tumours have a
mutated p53 gene. This scenario is illustrated in
Figure 3 and is consistent with the emerging
concept [21-251 that, in addition to inducing
apoptosis as an organism’s prctective effect, p53
may in some cases, help cells to recover from
toxic events by providing them temporary protection in GI arrest. We emphasize that in the case
of DENSPM, this concept has been validated
with cells that produce massive amounts of
SSAT. T h e generality of this effect remains to be
documented in other cell lines and with more
defined systems.
Portions of this work were supported by N.I.H. grants
R01-CA-22153, Institute Core Grant CA-16056 and
N.I.H. training grant CA-09072.
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Inhibition of voltage-activated K+ currents in cultured sensory neurons by the
polyamine spider toxin argiotoxin-636 may involve a polyamine transporter and an
intracellular site of action
R. H. Scott and M. T. Gibson
Department of Biomedical Science, Institute of Medical Science, Aberdeen University, Foresterhill,
Aberdeen AB25 2 2 0 , Scotland, U.K.
Introduction
Many predatory invertebrates inject and
immobilize their prey with venom. T h e venoms
of such predators contain toxins which act by
inhibiting the electrical excitability of neurons
and muscle cells. A predator’s venom may contain a cocktail of peptide and polyamine amide
toxins, and many of these toxins are inhibitors of
neurotransmitter and voltage-activated ion channels. Invertebrate toxins which act on ion channels are usually potent, producing functional
inhibition at doses which range from 1 0 n M to
1 pM, and they are often selective for a particular
type or subtype of ion channel. These toxins,
which inhibit ion channel activity, are of interest
because of their uses as lead compounds for the
design of pesticides. Additionally, toxins may
Abbreviations used: DRG, dorsal root ganglion;
MGBG, methylglyoxal bzs (guanyl-hydrazone);
NMDA, N-methyh-aspartate.
Volume 26
change the excitability of mammalian cells and
thus be useful pharmacological tools which may,
in the future, lead to novel therapeutic agents.
Argiotoxin-636 (Argiopine) is a polyamine
amide spider toxin which was originally isolated
from the venom of species of Argiope and Aruneus [1,2] including Aqzope lobutu, an African
orb web weaving spider. Argiotoxin-636 has also
been produced by total synthesis using a reduced
alkylation strategy [3]. In pharmacological
studies argiotoxin-636 was found to inhibit glutamatergic neuromuscular transmission in invertebrates. Consistent with this, argiotoxin-636 has
also been found to inhibit excitatory synaptic
transmission mediated by glutamate in rat hippocampal CA1 pyramidal neurons [4], and to have a
degree of selectivity for the N-methyl-D-aspartate
(NMDA)-sensitive glutamate receptor ion channel complex of vertebrates [S]. In addition,
argiotoxin-636 has also been used to characterize a-arnino-3-hydroxy-S-methyl-4-isoxazolepro-