EUEVIER
FEMS Microbiology
Letters 137 (1996) 123-128
MiniReview
Cell survival and multiplication
The overriding need for signals: from unicellular to multicellular
systems
L. Rasmussen a,*, S.T. Christensen
a, P. Schousboe a, D.N. Wheatley b
a Institute of Medical Biology, Department of Anatomy and Cell Biology, Odense University, Campuscej 55, DK-5230 Odense M, Denmark
b Cell Pathology Unit, Department of Pathology, Unil,ersity Medical School, Foresterhill, Aberdeen AB9 220, UK
Received 26 October
1995; revised 25 January
1996; accepted 29 January
1996
Abstract
There are clear similarities
in the control mechanisms for cell survival and multiplication in the two eukaryotes, the
ciliate Tetruhymena thermophilu and the yeast, Succharomyces cereuisiae. Cell multiplication in both organisms is activated
by the same compounds (phorbol esters, diacylglycerol, tetrapyrroles, etc.). These compounds also affect cell multiplication
and other activities in mammalian cell systems. This homology in control mechanisms in two distinct groups of unicellular
eukaryotes on the one hand, and in cells from multicellular animals on the other, leads us to propose that these cytoplasmic
control mechanisms for cell survival and multiplication originated in the unicellular eukaryotes.
Keywords: Tetruhymena thermophila; Saccharomyces cerecisiae; Escherichia coli; Signaling;
esters; Protoporphyrin
IX; Sodium nitroprusside; NO; Cyclic GMP; Evolution
1. Introduction
Signaling has mostly been studied in mammalian
systems, whereas it has tended to be overlooked in
unicellular eukaryotes. This has happened despite the
well-known fact that signals are involved in several
processes such as mating, chemotaxis and sporulation in single-cell organisms. Maybe it was tacitly
assumed that signaling
mechanisms
in uni- and
multi-cellular
organisms had very little in common.
However, signal systems originally
discovered in
mammalian cells exist in unicellular organisms with
* Corresponding
author. Fax: +45 65 93 04 80; Tel.: +45 66
15 86 96 ext. 2344; E-mail: [email protected]
0378-1097/96/$12.00
0 1996 Federation
PII SO378- 1097(96)00053-5
of European
Microbiological
Cell survival;
Cell multiplication;
Phorbol
high homology [l-3]. Here we discuss the view that
certain cytoplasmic signaling mechanisms connected
with activation of cell multiplication
originated in
free-living, unicellular eukaryotes and were later exploited and further developed by multicellular
animals. Studies into the unicellular eukaryotes may
show us signaling systems at work at an early point
in evolution.
At that time control systems were
relatively simple, because extensive integration between various mechanisms had yet to take place.
Cells have two major problems: they must survive
and multiply. Control of cell multiplication in multicellular eukaryotes is often initiated by first messenger molecules, and then sustained by secondary
messenger molecules and phosphorylation
cascades
[4]. The result is that “early genes” become actiSocieties. All rights reserved
vated [5]. Many cytoplasmic enzyme systems may be
involved in these processes, but what is the end-point
in the cytoplasmic
cascade‘? Here we show that
cyclic guanosine monophosphate (cGMP) is central
in the process that activates cell multiplication
in
both the unicellular
animal,
Tetr&wzenrt
thermophila, and in the yeast, Saccharom~ws cerel.isiae.
Fig
7.
Effects of various concentrations of human recombinant
insulin on cell survival and multiplication in cultures of Trtrtrhv-
2. Tetrahymena
wmt
rhrnnophikr
in conical
Cells of the free-living ciliate T. thermophila can
multiply to about lOh organisms per ml when grown
in a nutritionally
complete,
chemically
defined.
lipid-free nutrient medium. If the cells are thoroughly washed to dilute any “carry-over”
of stimulating compounds and transferred into conical flasks,
then the fate of the culture depends on the initial cell
density (Fig. I). At initial densities of 2500 cells per
ml, the cells double in number every 2-3 h, but at
250 initial cells per ml, they die rapidly (Fig. IA)
[6,7]. These cells at low initial density can be
“activated”
to multiply by different means [7-IO].
0
10
20
30
0
10
hours
Fig.
cally-
I.
Population density of Trtruhwnena
defined
20
30
hours
nutrient medium (CDM)
thrrmophilrr
in chemi-
in conical flasks as a
function of time and the initial cell density in the absence (A) and
presence (B) of cell-free conditioned medium (CCDM).
CCDM
was prepared from a culture having 50000 cells per ml and added
I:1 to fresh medium. Prior to inoculation, cells were washed in
either CDM
or in a TRIS/HCl-buffer
( > IO’-fold
dilution of the
original extracellular medium) to minimize carrying over of extracellular signals. (Redrawn from [6,7].)
in chemically-defined
flasks at 400
nutrient medium (CDM)
cells per ml in the presence (bold
rymbols) and absence (open symbols) of hemin (SO nM). Without
addition of insulin the cells died within IO h both in the presence
and ahaence of 50 nM hemin. The lines show results from those
cultures in which the cells multiplied and the numbers of hours in
lag phase before start of cell multiplication
(i.e. the higher the
points. the aborter the lag phases). (Redrawn from
[ 121.)
This can be done by cell-secreted compounds (Fig.
IB) or by compounds
activating cellular control
mechanisms.
Tetruhymenu
cells release activating
(autocrine) factors [7-91 but their identity is unknown. Three lines of evidence suggest that they
may be related to insulin-like material. First, insulin
added to Tetruhymena cultures at low inoculum densities rescues these cells from death at concentrations
as low as 10piJ M [ 1 I, 121. Second, Tetrahymena
cells release compounds with the same effects as
insulin on porcine adipocytes. These effects are inhibited by an antibody against insulin and by an
antibody against the porcine insulin receptor [ 131.
Third, Tetruhymena cells possess binding sites for
insulin [2], with similar saturation kinetics, temperature and pH dependencies
to those of mammalian
receptors [ 141. Interestingly,
insulin stimulates cell
survival and activates multiplication
in the ciliate at
two separate intervals, at nano- and again at low
pica- and femtomolar concentrations in cultures having low initial cell densities (Fig. 2) [ 121. Insulin
probably induces multiplication
per se rather than
glucose uptake in Tetrahymena, because this ciliate
reacts to insulin in the same way in a glucose-containing and glucose-free nutrient medium [ 1 I].
It is possible that insulin and the autocrine factors
activate protein kinase C and induce the formation of
cyclic guanosine
monophosphate
(cGMP). Three
“sets” of externally added compounds mimic their
actions. One set includes protein kinase C activating
L. Rasmussen et al./
FEMS Microbiology
compounds such as phorbol esters, oleylacetylglycerol, and certain lipids ([9,15], unpublished).
A second set includes hemin, protoporphyrin
IX, and
sodium nitroprusside
(an NO-releasing
compound)
[7,16,17]. They all play a role in activating a cytosolit, “soluble”, in contrast to a “particulate”,
membrane-bound
guanylate cyclase in mammalian cells
[ 181. Guanylate cyclase is a heme-containing
enzyme. The soluble iso-form of this enzyme is activated by protoporphyrin IX and by NO, which binds
to the heme group of the cyclase [ 181. The latter
enzyme is also activated by hemin in combination
with NO. In addition, sub-minimal concentrations of
hemin have additive effects together with both insulin (Fig. 2) [12] and sodium nitroprusside
[17].
Finally, the third set of compounds activating multiplication includes 8bromo cyclic GMP [ 171, a membrane-permeating
analogue of cGMP which is the
product of activated guanylate cyclase. These conclusions are supported by the findings that both
insulin and sodium nitroprusside (added singly) induced the formation of intracellular
cGMP in Tetruhymena [19]. Thus studies with activators indicate
two control mechanisms, one a protein kinase C, and
the other a guanylate cyclase, responsible for onset
of multiplication
in cultures of T, thermophiZu.
Results with inhibitors support this idea. Compounds inhibiting either protein kinase C or soluble
guanylate cyclase block cell multiplication
and induce cell death at any cell density. These compounds
include an inhibitor of protein kinases, staurosporine
[20], a competitive inhibitor of NO-synthase,
NCmethyl-L-arginine
[ 17,211, and the blocking agents of
the activity of soluble guanylate cyclase in mammalian cells [22,23], methylene blue and 6-anilino5,8_quinolinedione
([17], unpublished res.).
Protein kinase C is presumably
located “upstream” in relation to the soluble guanylate cyclase
(Fig. 3A). The evidence for this view is that the
inhibitory effects of staurosporine can be bypassed
by protoporphyrin IX or 8-bromo cGMP, but not by
phorbol esters, oleylacetylglycerol
or insulin (unpublished res.). Moreover, the effects of NC-methyl-Larginine are bypassed by an excess of arginine, or by
protoporphyrin IX, sodium nitroprusside, or 8-bromo
cGMP, but not by insulin (Fig. 3B) [17]. Of all these
compounds,
only 8-bromo cGMP circumvents
the
inhibitory effects of methylene blue and 6-anilino-
Letters 137 (1996) 123-128
125
A
Fig. 3. Schematic presentation
of putative signal transduction
mechanism pathways induced by autocrine factors required for
activation of cell multiplication in 7’etrahynena. (A) receptor +
protein kinase C + soluble (NO-dependent)
guanylate cyclase
system. (B) detail of the regulation of the soluble guanylate
cyclase system leading to formation of cyclic GMP. Abbreaiations: PM, plasma membrane;
PKC, protein kinase C; SGC,
soluble (NO-dependent)
guanylate cyclase; NOS. nitric oxide
synthase; NMA, NC-methyl-L-arginine;
t_-Arg, t_-arginine; SNP,
sodium nitroprusside; PPIX, protoporphyrin
IX; MB, methylene
blue; f., activation/inhibition.
5,8_quinolinedione
([17], unpublished res.). All these
results indicate cGMP as the last known compound
needed to effect transition from lag phase to active
cell multiplication.
We have recently suggested that Tetruhymena
cells can die by programmed cell death [6,20]. This
may happen either in the absence of their autocrine
factors (e.g. at low inoculum densities), or when
signal transduction via these factors has been blocked
126
L. Krrsrnussrrr ct trl. / FEM.5 Microhiolo~~
(e.g. by addition of the protein kinase inhibitor staurosporine). This cell death was postponed by inhibition of de nova protein synthesis. (Inhibition of de
nova RNA synthesis lowered the critical initial cell
density required for activation of cell multiplication
[6] in line with the notion that pr+rmed
mRNA
enabled the cells to keep producing the autocrine
factors.) The putative set of genes involved in this
cell death has still to be identified, but investigations
into cellular morphology. ultrastructure,
and DNA
patterns permit the suggestion that the staurosporine-treated cells die by a non-apoptotic
mechanism
ml.
3. Saccharomyces cerevisiue
S. cere~isiae cells also need to be “activated”
in
order to leave the lag phase and start multiplication
in a minimal synthetic medium with glycerol as the
carbon source [24.25]. This activation was brought
about by glucose, 6-deoxyglucose,
hemin, protoporphyrin IX (all added singly), or by Ca’+ together
with the ionophore A 23187 [24.25]. Furthermore.
cell multiplication
is blocked by 6-anilino-5,8quinolinedione,
but this inhibitory effect was abolished by 8-bromo cGMP (unpublished res.). Again.
this indicates cGMP as the last known compound
needed to effect transition from lag phase to multiplication in S. cerecisiae.
The phorbol esters and oleylacetylglycerol,
i.e.
the protein kinase C activators. had no activating
effect by themselves, but when either of them was
given together with protoporphyrin IX, the doubling
time of the cells was reduced [25].
4. Prokaryotes
Work in our laboratories (DNW) is currently appraising the situation with regard to bacteria (E.
co/i). The work is largely done in a comparative
manner because it has already been recognized that
prokaryotes may indeed need signals, especially under poor nutritional conditions, to multiply from low
densities [26,27]. The evidence from Fuqua et al.
[28] also shows that the marine bacterium Vibrio
fischeri signals through a light-emitting
mechanism
Letter.\ 137
f IYY6)12%
12X
when in crowded conditions through activation of its
lux genes, which is reduced in diluted cultures.
External signals may not be essential in E. coli. but
the need for some kind of “pump-priming”
signaling of an autocrine nature cannot be ruled out,
especially from cell densities of less than I cell per
ml. Stimulants accelerate the rate at which multiplication gets under way and can dramatically reduce
the lag phase. Some stimulants correspond with those
which work in eukaryotes; others do not, and these
may be peculiar to bacteria. For example, insulin can
stimulate certain bacteria [2] at low concentrations in
a similar manner to Tetrahymena, as can nitroprusside in our recent experiments, whereas homoserine
lactone derivatives [29,30] have no effect on the
ciliate (unpublished
res.1. The effect of 8-bromo
cGMP is currently under examination in E. coli.
5. Conclusions
People growing cells have always known that
cultures having low cell densities had difficulties in
. ‘taking’ ’ . With model cells from various groups,
prokaryotes, unicellular animals and yeasts, we begin
to get insight into intracellular mechanisms activating cell multiplication
after subcultivation
in previously formulated media. Here we limit our discussion mainly to the eukaryotes.
First, results obtained with T. thermophila lead us
to suggest that first messengers for activation of cell
multiplication exist in this organism. It is not known
whether they are also present in the yeast cells.
Second, results from T. thermophila and S. cere~~isiae further indicate a remarkable homology in
cytoplasmic events leading to activation of cell multiplication. In both cases guanylate cyclase and cGMP
seem to play important roles, and it is indeed an
intriguing
possibility that this homology includes
prokaryotes.
We assume that these common survival and multiplication controls in the eukaryotes evolved in the
group of free-living unicellular
organisms. All of
these lead a so-called “feast-or-famine”
existence:
they sometimes have an abundance of food, but they
also encounter long periods of starvation [31]. During evolution, those cells possessing the most efficient mechanisms for closing down metabolism and
L. Rasmussen et a/. / FEMS Microbiology Letters 137 (1996) 123-128
signal-induced
multiplication
until food reappeared
also had the best chances of surviving. The same
control mechanisms, or very similar ones which have
evolved from them, operate in cells from multicellular animals.
Biologists in general have learned much about
control systems in cells from multicellular
eukaryotes with regard to their balance between survival,
multiplication
and differentiation,
often from growing them in relative isolation in culture. We have,
however, been slow to appreciate that unicellular
organisms such as yeast and protozoa already possessed very similar mechanisms [l-3,17,25].
Indeed,
work on the flagellate Trypunosomu cruzi showed
that cells can die by programmed
cell death and
apoptosis, probably as a consequence of the absence
of autocrine factors [32]. Work with unicellular eukaryotes provides cleaner (weaker noise-to-signal
ratios) and less ambiguous answers to the problem of
the need for signaling for fundamental
processes
such as cell survival and multiplication.
In agreement with Raff [33], we suggest that in
many cases (Raff claims all), eukaryote cells do not
survive in the absence of such signals despite the
presence of sufficient “raw materials” being available to support multiplication
to high densities. But
signals to survive and multiply probably represent
only a fraction of the total spectrum of messages
they must send to one another for a variety of other
activities. In line with the ideas of Ameisen et al.
[32] and Vallesi et al. [34], it would seem that
signaling in unicellular
organisms is important in
many cell activities, being necessary for cell survival
and multiplication
[9,12,17,32,34-361,
chemotaxis,
conjugation
and differentiation
[32,34,37-401.
Finally, signaling may also be implicated in cell suicide and programmed cell death [6,20,30,41].
Acknowledgements
We thank Professor Martin Raff, University College, London, and Dr. Jargen Friis, Odense University, for helpful comments. Our studies were supported by funds from the Danish Natural Science
Research Foundation (11-1088-l),
the Carlsberg and
the NOVO Foundations, Copenhagen, Denmark.
127
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