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Biochemistry of the cell cycle

Article in Biochemical Journal · April 1987

DOI: 10.1042/bj2420313 · Source: PubMed

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Biochem. J. (1987) 242, 313-321 (Printed in Great Britain) 313

REVIEW ARTICLE
Biochemistry of the cell cycle
David LLOYD
Department of Microbiology, University College, Newport Road, Cardiff CF2 ITA, Wales, U.K.

Cellular organization: temporal aspects tives of sub-molecular motions (Williams, 1981), metab-
Those processes that constitute the cell cycle include all olism (Reich & Sel'kov, 1981), and growth processes
the biosynthetic mechanisms involved in growth, the (Lloyd et al., 1982b) are now becoming clear. Functions
provision of energy, the development of extending and structures differ only in the time constants of their
membranes and surface structures, the genesis of new component reactions. To study the rich complexities of
organelles, and the mechanics of segregation of compo- function and formation that occur during cellular growth
nents at cell division. As well as elucidating the stages of and division, it is necessary to examine the processes
the processes themselves, the spatial and temporal either in single cells or in a population composed of
control of this developmental cycle needs to be individuals all at the same stage of cell cycle progression.
understood. Were the synthesis of new constituents to The former approach is constrained by current limita-
occur continuously from one cell division to the next, tions of sensitivity and applicability of non-invasive
then the cell would be biochemically identical at all times methods to single cells. Even so, many of the most
in its life time: individual molecular components would fundamental questions must eventually be tackled at this
be always at a fixed stoichiometry. Classically, biochem- level, and rapid advances (e.g. in digital scanning
istry has been studied as if this were the case. Thus most fluorescence microscopy techniques; Taylor et al., 1986)
studies of growth, even those performed with micro- suggest the imminent flourishing of single-cell research.
organisms, neglect entirely the concept of discontinuity: The great attraction of this avenue is the emphasis that
the growth of individual cells and increase in cell it will place on the differences between individual cells,
numbers are lumped together in a measurement of and on the heterogeneity in a population even when
increased optical density or dry weight ('biomass'). Such cohorts of identical ages are taken. The extent of this
global measurements ignore the differences between natural variation in individual cells during their normal
individuals: information obtained from the population development merits investigation. In contrast, most
gives only time-averaged values over an interval equal to current biochemical measurements made on a sampled
the cell division time. The fine details of sequenced events population assume homogeneity; the expression of such
are blurred and lost (Edwards, 1981; Lloyd et al., 1982b). measurements as a specific activity based on cell
numbers implies a touching faith that an equivalent
contribution is derived from each individual.
Methods for study of the cell cycle Apart from microscopy-based measurements there are
The time span of the cell division cycle may be as short other methods for examining the heterogeneity of cell
as 9.8 min in the marine bacterium Vibrio natriegens populations. Thus, the use of flow cytometry and flow
(Eagon, 1962). It may be about 24 h in proliferating cytofluorimetry in biochemistry has become routine,
animal or plant tissues or in micro-organisms in natural especially in laboratories working with cultured mam-
ecosystems. In some cases, a quiescent stage lasting years malian cell lines (Bohmer, 1982). Most often used for
may eventually lead to a normal cycling state or a cell cycle analysis by fluorimetric DNA measurement,
malignant tumour growth (Baserga, 1985; Epifanova & these techniques now find varied application, especially
Polunovs, 1986). The importance of polypeptide growth where cellular heterogeneity can be resolved by using
factors and their receptors in growth control has been fluorescent monoclonal antibody staining.
reviewed recently (James & Bradshaw, 1984). Thus, even The second more conventional approach to the study
in the shortest of cell cycle times, metabolic events will of the cell cycle is to prepare a synchronous culture, in
have occurred by their thousands, those membrane- which all the cells are in phase; the whole culture then
associated phenomena not limited by diffusion may be behaves as a coherent population. Several practical
counted in millions, and vibrational events within solutions to the difficulty of synchronizing populations
proteins in hundreds of millions (Fig. 1). Although strict are available, and these methods yield sufficient amplifi-
ordering of processes and events on a nanosecond time cation to allow conventional biochemical analyses.
scale may not directly determine the course of the cell However, caution must be exercised, and it must be
cycle, all these subsystems contribute in the hierarchy of admitted that many of the published observations
temporal controls necessary for progression from one obtained by the use of synchronous cultures are
cell division to the next. controversial (Lloyd et al., 1982b). This is because many
On a longer time scale, the cell division cycle is itself investigators have failed to take adequate precautions to
often dominated by physiological adaptation to tidal or enable distinction between those phenomena which are
circadian controls (Sweeney, 1982), just two examples of real cell cycle events from those artifacts which result
evolutionary matching to geophysical cycles. Until from the experimental procedures necessary to obtain
recently the biochemist's preoccupation with the struc- synchrony. This problem is not confined to the data in
tural hierarchy of living systems had sometimes led to a the older literature which relied on induction synchrony.
neglect of temporal relationships. The dynamic perspec- It is now realized that it is difficult (and perhaps not
Vol. 242
314 D. Lloyd

Cell cycles of:

Cultured
mammalian cells

Activation
of Na+ channel Tetrahymena
Events and processes Saccharomyces
within proteins
Rotation of Vibrio
rhodopsin in rod
membrane Complete cycle of Wall and
muscle crossbridging membrane
assembly __
Backbone
local motions Transcription,
Fastest enzyme Complete cycle of Na' K+ translation
!.Ridlp ihain I reaction pump t
.
.lua
local motion4
r
j l Intermediary metabolism 0 0 ...

Transit V Energy conservation and Circadian Lunar Annual Human

$ time of Na+ in membrane transport phenomena life
channel span
| Protein and membrane
+--- conformational changes
ti reoxidation of electron transport chain
CH3 rotations
Atomic Cytochrome a + a3 Succinate dehydrogenase
collisions
le -^------,H20in rotation
bulk
water
I I l I
-12 -6 6
log [Time (s)]
Fig. 1. The time domains of lving systems

possible) to avoid perturbative influences completely; value of F would be unity, whereas for an exponentially
rather few methods for the preparation of synchronous growing culture its value would be zero. Values greater
cultures can claim to be minimally perturbing, and even than 0.85 suggest that induction plays a role in
for these it is always necessary to present as controls data synchronization; those between 0.6 and 0.85 are
from identically treated asynchronous cultures. The regarded as satisfactory for selection synchrony.
methods of choice involve selection of similarly-sized (ii) The cell cycle time of the synchronous culture
organisms by a short, low-speed centrifugation (e.g. should be similar to the mean generation time of the
12 ga., 1 min) of the culture in tubes (Chagla & exponentially growing culture from which it was derived.
Griffiths, 1978) or in a continuous-flow rotor (Lloyd Deviation indicates perturbation.
et al., 1975; Edwards & Jones, 1977; Lloyd, 1983). These (iii) Events should be repeated in successive cycles.
simple methods use minimal physical disturbance, are Often the first cell cycle is distorted by the experimental
faster and simpler than others currently employed (see procedure, especially over the first hour or so. Synchrony
Lloyd et al., 1982b, for experimental details), and have in successive cycles decays, mainly because of the natural
the other great advantage that organisms never lose sight dispersion of cell cycle times. Thus, even when
of their growth medium. They thus never become population growth rate is constant, individual cells have
transiently deprived of nutrients or 02 widely differing cycle times, the coefficient of variation
Criteria for assessment of the success of a cell frequently being of the order of 15-20% (Brooks, 1985).
synchrony procedure include the following. Where feasible, it is an excellent practice to compare
(i) Measurement of a synchrony index, e.g. that of results from different synchronizing techniques (e.g.
Blumenthal & Zahler (1962) given by: Knacker et al., 1985). In those few cases where this has
been done, large discrepancies due to perturbation effects
F = (Nt/NO-20L/fl) are usually revealed when induction methods have been
employed. Even light-dark synchronized cell division
where Nt is the number of organisms after division is commonly used for studies of the cell cycles of
completed, No the number of organisms before division, phototrophs (Adams et al., 1984) must be regarded as
a is the time taken for the organisms to divide and , is an induction method, and separation of the events of the
the generation time (Fig. 2). For a culture showing cell cycle from those of light-dark adaptation processes
instantaneous and exact doubling in cell numbers, the is not straightforward.
19g87
Biochemistry of the cell cycle 315

examples are those that control or execute specific cell

oI.I= cycle processes. Hartwell's (1974) definition which
confines interest to those mutations that result in arrest
of the cell cycle at a specific phase may however be
unduly restrictive. There are mutants that lack a major
portion of the cell cycle (Liskay, 1977) and some that
arrest at two distinct stages. A very successful and
E complete investigation of the yeast cdc mutants has
zn enabled the elucidation of the order and interdependence
E of cell cycle processes (Hartwell, 1978). Those cell cycle
._
co mutants which divide at a size very different from that at
o which the wild type divides [whi mutants of Saccharo-
myces cerevisiae (Sudbery et al., 1980) and wee mutants
E of Schizosaccharomyces pombe (Nurse, 1981)] have
=
proved valuable systems for probing 'sizer' control
mechanisms.
cr Despite the burgeoning interest in mammalian cell
cycle mutants (Table 2) few of the lesions responsible are
biochemically defined. Exceptions to this are seen for
ts A159, a topoisomerase II mutant (Colwill & Sheinin,
1982, 1983), and for ts 85 (Mita et al., 1980; Matsumoto
et al., 1983). Surprisingly this latter mutant which arrests
Time on G2 is defective in the ubiquitin pathway. This
Fig. 2. Assessment of the degree of synchrony in a population of discovery points to an unexpectedly crucial role of this
cells or organisms: the synchrony index of Blumenthal & pathway at a particular stage of the G2 phase.
Zahler (1962) In the case of the yeast cell cycle, examples where
For explanation see the text. specific biochemical lesions have been pinpointed are
given in Table 3. It is remarkable that some of these
mutations should give phenotypes falling within the
definition of a cell cycle mutation, e.g. cdc19, which
has a temperature-sensitive pyruvate kinase (Kawasaki,
Biochemical changes during the cell cycle 1979). It may be that this enzyme, apart from its key
Table 1 shows some examples of changes in the levels glycolytic role in energy generation, has a second as yet
of cell constituents or processes during the cell cycle. unrecognized function. The application of 13C-n.m.r.
These are taken from reports of the last year. For a spectroscopy in vivo presents a powerful approach to the
summary of earlier work see Lloyd et al. (1982b). rapid and unequivocal localization of metabolic defects
(Gilles & Benoit, 1983); similar studies using phos-
Cell cycle mutants phorus or nitrogen n.m.r. offer other rapid diagnoses.
Isolation of cell cycle mutants and their characteriza- Cloning of CDC genes affords another method of
tion represents a major part of current endeavours; as identification, e.g. the primary structure homology
most of these mutations are lethal they must be isolated between the CDC28 gene product of S. cerevisiae and
as conditional (usually heat-sensitive) mutants. Products bovine cyclic AMP-dependent protein kinase (Lorincz &
of thousands of genes are required for the normal growth Reed, 1984).
and reproduction of cells, but the most interesting Cell cycle mutations in several prokaryotes have been

Table 1. Constituents and processes during the cell cycle: some recent (1986) studies

Constituent process Organism(s) Reference

DNA replication Eukaryotes Campbell (1986)

DNA ligase expression Saccharomyces cerevisiae White et al. (1986)
Schizosaccharomyces pombe a
Human histone gene Mouse cell Green et al. (1986)
expression
Histone and tubulin gene Physarum polycephalum Carrino & Laffier (1986),
expression Laffler & Carrino (1986)
a-Tubulin genes Schizosarcharomyces pombe Adachi et al. (1986)
Microtubule dynamics PTK-2 cells Debraban et al. (1986)
Diadenosine tetraphosphate, Physarum polycephalum Garrison et al. (1986)
adenosine tetraphosphoguanosine
Calmodulin levels HL-60 Yen et al. (1986)
Spindle pole body duplication Saccharomyces cerevisiae Baum et al. (1986)
Protein phosphorylation Saccharomyces cerevisiae Tripp et al. (1986)
Protein modification Early mouse embryo Howlett (1986)
Chromatophore membranes Rhodospirillum rubrum Myers & Collins (1986)
Vol. 242
316 D. Lloyd

Table 2. Cell cycle mutants derived from mammalian cell lines

Organism Cell line Mutant Arrest point Reference

Chinese hamster WglA K18, K27, Late G1; entry Melero (1979)
K33, 4/3, into S
4/2, 3/1
WgIA tsK/34C G, Tenner et al. (1977),
Otsuka & Scheffier (1978),
Landy-Otsuka & Scheffler (1978)
WglA H3.5 G, Landy-Otsuka & Scheffler (1980)
CCL39 BF113 G1 after one cycle Scheffler & Buttin (1973)
CHO MSl-l Cytokinesis Thompson & Lindl (1976)
CHO tsC8 DNA synthesis McCracken (1982)
CHO tsD, ts224 Various Okinaka & Barnhart (1978)
and others
CHO* cs4-D3 GO/Gl Crane & Thomas (1976),
(reversible) Crane et al. (1977),
Berger et al. (1979),
Lomniczi et al. (1977)
CHO* CRRE5 G1 Ling (1977)
GM75 TSl Cytokinesis Hatzfeld & Buttin (1975)
D6 (pseudo-diploid) ts-l GI Hori (1977)
CCL61 1129 and 1132 Reversibly in G, Ohlsson-Wilhelm et al.
and less (1976 1979)
reversibly in G2
CHO-K1 1 C3 Late S/G2 Marunouchi & Nakano (1980)
CHL-V79 Gl+-4, G, Liskay & Prescott (1978)
Gl+-5
CHO tsAMAR-1 G, Ingles (1978)
Syrian hamster BHK NW1 Cytokinesis Smith & Wigglesworth (1972)
BHK dna-tsBN2 DNA synthesis Eilen et al. (1980)
BHK tsBN75 G2 and S Nishimoto et al. (1980)
BHK ts422E G2 and mitosis Mora et al. (1980)
BHK21/13 tsAf8, Early to mid Meiss & Basilico (1972),
tsll, tsl3 G1 Burstin et al. (1974),
Burstin & Basilico (1975),
Kane et al. (1976),
Ming et al. (1976),
Talavera et al. (1976),
Liskay & Meiss (1977),
Talavera & Basilico (1977),
Chang & Baserga (1977),
Moser & Meiss (1975, 1977)
Floros et al. (1978),
Rossini & Baserga (1978),
Jonak & Baserga (1979),
Rossini et al. (1979, 1980)
BHK211 tsHJ4 Late G1, early Talavera & Basilico (1977)
S function
BHK21 tsBTN-1 S? Nishimoto & Basilico (1978),
tsBN-2 G, and S Yanagi et al. (1978),
tsBN75 S Nishimoto et al. (1980)
BHK21 ts422E Prior to division Toniolo et al. (1973),
cells have 4n Mora et al. (1980)
amount of DNA
and very large
nuclei
Hamster HM-1 ts-546 Mitosis Wang (1974),
(metaphase) Wang & Yin (1976)
HM-1 ts-655 Mitosis Wang (1976)
(prophase)
HM-1 ts694, ts559 GI Chen & Wang (1977)
HM-1 ts-550c G1 and G2 Chen & Wang (1977, 1982)
HM-1 ts687 Mitosis Wissinger & Wang (1978)
Mouse CAK B54 Late G, Liskay & Meiss (1977),
Liskay (1974),
Farber & Liskay (1974)
L tsA lS9 S Thompson et al. (1970, 1971),
Sheinin (1976a,b),
Setterfield et al. (1978),
Sheinin & Gutman (1977),
1987
Biochemistry of the cell cycle 317

Table 2. (continued)

Organism Cell line Mutant Arrest point Reference

Sheinin et al. (1978),
Colwill & Sheinin (1982, 1983)
L tscl Late S or G2 Setterfield et al. (1978),
Sheinin & Gutman (1977),
Thompson et al. (1971)
Balb/3T3 A8, A83 and G1 Naha et al. (1975),
5 others Naha (1979a,b),
Naha & Sorrentino (1980)
Balb/c 3T3 ts-2 S Slater & Ozer (1976)
FM3A ts85 Late S or G2 Mita et al. (1980)
FM3A tscl.B59 Cytokinesis Yasuda et al. (1981),
Nakamo et al. (1978)
FM3A tsT244 DNA synthesis Tsai et al. (1979)
FM3A tsl3lb DNA synthesis Hyodo & Suzuti (1982)
Murine leukaemic L51787 ts2 Mitosis and Shiomi & Sato (1976, 1978),
ts39 cytokinesis Sato & Hama-Inaba (1978)
Rat 3Y1 tsD123 G, Zaitsu & Kimura (1984)
African green BSC-1 ts3,ts5,ts9 S Naha (1973)
*
Cold-sensitive.

described: these provide fundamental insights into theory (Goodwin, 1980) and the participating of the ion
mechanisms of chromosome replication, growth and cell currents observed by Jaffe (1981). Further information on
division (e.g. in Escherichia coli; Donachie, 1981) and of methods of isolation and characteristics of cell division
morphological differentiation (e.g. in Caulobacter cres- cycle mutants is to be found in Lloyd et al. (1982b).
centus; Shapiro, 1976). Amongst eukaryotic cells other
than yeasts (and those of mammalian origins) special The control of cell division
mention must be made of three systems. In Aspergillus In rapidly growing populations, cells progress from
nidulans the mitotic mutants isolated by Morris (1980) mitosis to S-phase and on to mitosis again with a
and coworkers identify the time structure of nuclear regularity that has encouraged the idea of a cycle of
events and the interactions of nuclear envelope, spindle events. Close to this idea is that of a biochemical
pole bodies, kinetochore and microtubules during oscillator mechanism, and it is not surprising that many
chromosome segregation. In Chlamydomonas reinhardii proposals for cell cycle control are based on limit cycle
genetic dissection of the stages of organelle development models. Sel'kov (1970) suggested synthesis and degrada-
was pioneered by Howell (1974). Finally, the production tion of thiols and disulphides, oxidation and reduction
of a wide variety of morphologically aberrant mutants of providing the control variables. Gilbert (1981) developed
the ciliate protozoon Tetrahymena pyriformis (Frankel a theory based on this type of control system, and indeed
et al., 1980) provides a system suitable for the as explained by Kauffman & Wille (1975), a limit cycle
investigation of the basic mechanisms of the generation mechanism, in which mitosis is triggered when an active
of form. The mutant protozoa provide systems suitable substance reaches a critical threshold concentration, can
for testing new hypotheses, e.g. the morphogenetic field mimic many experimental observations. For instance the

Table 3. Biochemical lesions in cdc mutants in yeasts

Organism Mutant Defective enzyme Reference

Saccharomyces cdc7 Protein kinase Patterson et al. (1986)

cerevisiae
cdc8 Thymidylate kinase Jong et al. (1984),
Sclafani & Fangman (1984)
cdc9 DNA ligase Johnston & Nasmyth (1978)
cdcl9 Pyruvate kinase Kawasaki (1979)
cdc2l Thymidylate synthetase Game (1976)
cdc3O Phosphoglucose isomerase Dickinson & Williams (1987)
cyri Adenylate cyclase
bcyl Cyclic AMP-dependent Matsumoto et al. (1983)
protein kinase
(regulatory subunit)
Schizosaccharomyces cdc2 + Protein kinase Simanis & Nurse (1986)
pombe cdcl 7 DNA ligase Nasmyth (1977)
cdc22 Nucleoside diphosphokinase Dickinson (1981)
Vol. 242
318 D. Lloyd

'set-backs' in cell cycle progression produced by heat

shocks (Zeuthen & Scherbaum, 1954; Brewer & Rusch,
1968), or pulses of protein synthesis inhibitors, point to
the assembly of a thermolabile protein-containing
structure necessary for cell division. Whether the
oscillator is a continuous one or is of the relaxation type
is still debated (Tyson & Sachsenmaier, 1978).
Another viewpoint emphasizes the variability of cell
cycles rather than their regularity (Brooks, 1985). The
basis of the variability of the duration of the G1 phase
suggests that commitment to DNA replication and
mitosis in mammalian cells might occur at random with
constant probability per unit time (Cattaneo et al., 1961;
Burns & Tannock, 1970; Smith & Martin, 1973). Much
of the evidence presented for this 'single transition-
probability' model can be used to promote alternatives
[e.g. the 'G1 rate' model (Castor, 1980), the 'sloppy size
control' model of Lord & Wheals (1984) or even the
'oscillator' model (Gilbert, 1982)].
Finally we have the 'dependent pathways' model in
which one or more causal sequences finally join to give
a closed loop of states. Order and interdependence of
events in the cell cycle of S. cerevisiae clearly emerges
Cell cycle time
from the work of Hartwell (1978). Identification of a key
regulatory step in the G1 phase ('start') mediated by the
Fig. 3. Timing provided by the temperature-compensated period gene product of gene cdc28 initiates the cell cycle
of the ultradian clock together with 'sizing' provided by provided that carbon and nitrogen sources, sulphur,
a temperature-independent threshold concentration of phosphorus and required amino acids are all present
some constituent may determine cell cycle time (Hartwell, 1974; Johnston et al., 1977; Shilo et al., 1978)
This has discrete values; i.e., as temperature is decreased, and the mating pheromone is absent (Bucking-Throm
cell cycle times increase by quantal increments (Lloyd & et al., 1973). Nutritional shift-up experiments (Carter
Kippert, 1987). et al., 1978) and a plethora of other data are consistent

U LTRAD IAN
INPUTS CLOCK OUTPUTS
(Perturbative influences) (Overt ultradian rhythms)

Heat shocks Respiratory rhythms

Serum pulses w*Adenylate pool sizes
-. Protein accumulation
Ionizing radiation
U.v. radiation
Motility rhythms
Quantized cell cycle times
Fig. 4. Ultradian clock control of cellular processes
The period of the clock (generally within the range 0.5-4 h) differs from one organism to another.

Table 4. Some recent (1986) cell cycle studies with medical implications

Subject Reference

Development of thermotolerance during hyperthermia Holahan & Dewey (1986)

Factors controlling the B-cell cycle Melchers & Andersson (1986)
Phorbol esters and interleukin-1 activation of T-cells Davis & Lipsky (1986)
Interleukin-3 and cell cycle progression Kelvin et al. (1986)
Cell cycle specific effects of anticancer drugs Kimmel & Traganos (1986)
Bone marrow cell cycle kinetics and thymic peptides Tucker et al. (1986)
Adenosine and the cell cycle of human lymphoid-cell Vanderkaan et al. (1986)
lines
Suppression of tumorigenicity by cell cycle control Wille & Scott (1986)
Oestrogen stimulation and anti-oestrogen inhibition of Lykkesfeldt et al. (1986)
human breast cancer cell lines
Melanoma-associated antigen expression in human Kameyama et al. (1986)
melanoma cell lines
Regulation of epidermal growth factor receptors by Fanger et al. (1986)
glucocorticoids in HeLa cell lines
1987
Biochemistry of the cell cycle 319

with the idea that initiation of the cell cycle at 'start' is Burstin, S. J. & Basilico, C. (1975) Proc. Natl. Acad. Sci.
followed by a constant time period before attainment of U.S.A. 72, 2540-2544
a critical size (mandatory for initiation of budding). Sizer Burstin, S. J., Meiss, H. K. & Basilico, C. (1974) J. Gen.
and timer controls may involve genes (e.g. weel and cdc2) Physiol. 84, 397-408
in Schiz. pombe in controls that monitor cell mass and Campbell, J. L. (1986) Annu. Rev. Biochem. 55, 733-771
Carrino, J. J. & Laffler, T. G. (1986) J. Cell Biol. 102,
determine mitotic timing (Nurse, 1981). 1666-1670
Attempts to reconcile these disparate models (Ed- Carter, B. L. A., Lorincz, A. & Johnston, G. C. (1978) J. Gen.
munds, 1984) of cell cycle controls may combine timing Microbiol. 106, 221-225
by a limit cycle oscillator with a size threshold (Shymko Castor, L. N. (1980) Nature (London) 287, 857-859
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mechanisms is that no definite molecular candidates are (London) 190, 923-924
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the oscillator corresponds to the cell cycle time it will be Chang, H. L. & Baserga, R. (1977) J. Gen. Physiol. 92, 333-344
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Chen, D. J. & Wang, R. J. (1982) Som. Cell Genet. 8, 653-666
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