Szabad, Biology booklet 22.
22. THE CELL CYCLE.
Cell size control. Cell cycle phases. Check points and
control factors. The chromosome cycle. Replication.
Cytoplasm and centrosome cycles. Yeast species and
the cdc mutations. Cyclines and cyclin-dependent-
protein kinases. Cell senescence.
INTRODUCTION
Cells of the multicellular organisms originate through
well-coordinated cell cycle control mechanisms. In
every second, there are several millions of cells
dividing in a human body. The daughter cells are
reminiscent of the mother cell: they have the same
number of chromosomes, one centrosome upon
mitosis, and usually share equally cytoplasm of the
mother cell. During cell proliferation cells repeat the
same events over and over again in a cycle fashion.
What determines cell size? How and when do cells
divide? How do cells double their chromosome
content and distribute equally between the daughter
cells? What types of molecular factors ensure cyclic
progression of cellular events? How can we get to
know cell cycle regulating components? How many
times can cells divide? Are there connections among
cell cycle progression and aging? The present
chapter aims to answer the above questions.
Connection between cell size and the initiation of
cell proliferation
Fig. 22.1. Reaching maximum size, cells prepare to
divide. Initiation of the cell division program
depends on cell size rather than on nutrient supply.
It has been well established that cells grow in size
following mitosis (Fig. 22.1). Cells will initiate
division upon reaching maximum size. Mitosis
initiation is determined on the basis of cell size and
not by the supply of nutrients (Fig. 22.1). (There are
exceptions, however. During the so-called cleavage
cycles of embryogenesis cells divide without
reaching maximum size and hence smaller and
smaller cells originate.) How do cells know the size
they need to reach before they start to divide?
Although the exact mechanism is not known, it
appears that there is a constitutively expressed gene,
let's say the A gene, and its gene product represses
expression of the B gene. Product of the B gene is
required to initiate cell division. Along growing of
the cell, concentration of the A gene product
gradually decreases. (After all, protein molecules do
not live forever. They are destroyed by the so-called
proteasomes, the protein digesting machinery in
the cells.) Once the A gene product concentration
drops below threshold level, its repression effect is
eliminated and hence the B gene is free to be
expressed. Cell division will initiate following the
expression of the B gene. The proposed model well
may be correct, since for example, hind brain of a
haploid salamander contains many more small cells
than hind brain of a tetraploid salamander with large
cells in its brain (Fig. 22.2).
Fig. 22.2. Cross-section of the hindbrain of haploid
(A) and a tetraploid (B) salamander.
Phases of the cell cycle
Cell cycle implies successive steps of divisions and
preparation for cell divisions. During the successive
cell cycles events happen is a precisely fixed order
along life of the cells. The period between two
mitoses is called interphase (Fig. 22.3). Perhaps the
so-called synthesis (S) phase of interphase is of most
importance: replication of DNA molecules takes
place during S phase and sister chromatids form. (See
also Chapter 5.) M and S phases are separated by G
(gap) phases: G1 between M and S, and G2 between
S and M (Fig. 22.3). G1 lasts sometimes for a very
long period of time, as in the neurons. The long G1s
are called G0. (We mention that there are special
types of divisions, e.g. the so-called cleavage
divisions during early frog embryogenesis, in which
the cell cycle contains only M and S phases.)
 Szabad, Biology booklet 22. 2

Cell fusion experiments. SPA, re-replication

block, MDF and MPF
Existence of cell cycle phases was revealed in cell
fusion experiments. In cell fusion experiments
synchronized cell cultures were used, in which every
cell was in the same phase of the cell cycle. The
simplest way of synchronizing cells is probably the
so-called mitotic sake off technique (Fig. 22.6).
Mitotic cells round up and do not attach firmly to the
culture vessel. The interphase cells on the other hand,
are firmly fixed to the tissue culture flask. Once
lengths of the cell cycle phases were established,
cells in the desired phase of the cell cycles can be
prepared conveniently.

Fig. 22.3. Phases of the cell cycle. The interphase

between two M phases (mitoses) is divided into G1, S
and G2 phases. The G1/S and the G2/M transitions are
important checkpoints in cell cycle control.

Duration of the cell cycle phases was determined as

follows (Fig. 22.4). A brief pulse of 3H-labeled
thymidine (to specifically label DNA) was added to
an asynchronously proliferating population of cells in
a cell culture. 3H-thymidine will be incorporated into
the replicating DNA and thus all the S phase cells
will be labeled. Let s take cell samples following
different amount of time following the pulse and
prepare autoradiograms of the cells. To interpret the
results it is helpful to depict the cells as though they
were distributed on a turntable, all moving around the Fig. 22.5. Cells in the M phase of the cell cycle
cycle at the same rate and with the position of each round up and can be shaken off from the plastic
cell on the turntable reflecting the stage it has surface of the culture flask.
reached in the cycle (Fig. 22.4). After a time equal to
the length of G2, the labeled cells begin to enter M
phase. After a time period equal to G2 + M, all cells Following fusion of G1 and S phase cells, the one in
in M are labeled, and so on. By waiting for the label G1 enters S phase, showing the presence of an S-
to appear in M phase, to progress beyond it, and then phase activator (SPA) in cytoplasm of the S phase
to reappear in M phase, one can establish the cell. SPA is required to initiate S phase. Under
duration of G2, M, S, G1 and the entire cell cycle. normal circumstances the G1 cells wait at the G1/S
They are about 3, 1, 7, 11 and add to roughly 24 phase border as long as there is no SPA in their
hours. Length of especially the G1 phase may change
significantly.

Fig. 22.4. Method to establish length of the cell cytoplasm. Behavior of the G1+S cell fusion
cycle phases. experiments clearly showed the importance of the

G1/S point in regulation of cell cycle progression.
G1/S is called restriction point (start in yeast) since
it provides an opportunity to stop or start cell cycle
progression (Fig. 22.3). Cells that passed the G1/S
checkpoint will accomplish S and G2 phases and
reach to G2/M, the other checkpoint in cell cycle
progression control.
It is rather peculiar that following fusion of an S
and a G2 cell, the one in G2 remains in G2 and does
not enter S phase. Apparently SPA does not act on
the G2 cells. Results of the G2 + S fusion experiments
clearly showed the presence of a chromosome-
associated factor that prevents repeated replication of
the chromosomes in the G2 cells. The mechanism is
called re-replication block and implies that
chromosomes replicate once and only once during
the S phase of the cell cycle. Another important
observation of the G2 + S fusion experiments is that
the G2 cell will wait for the S partner catching up.
When both components are in G2 at the G2/M
checkpoint, the two partner cells enter M together.
The former observation shows the presence of a
cytoplasmic factor, mitosis delaying factor, MDF,
that prevents entering the M phase before completion
of replication and arranging the chromatin the proper
ways (Figs. 22.3 and 22.6).
Fig. 22.6. Fate of partner cells following fusion of
cells in different stages of the cell cycle.
When M phase cells were fused with cells in any
other phase of the cell cycle, cells in G1, S or G2
entered M phase in minutes (Fig. 22.6). The factor
present in cytoplasm of the M cells is MPF, mitosis
promoting factor. MPF was formerly called
maturation promoting factor since it induced
maturation of the primary oocytes of the frog
Xenopus laevis (Fig. 22. 7). In Xenopus laevis, the
primary oocytes are arrested in the G2 phase of the
first meiotic division (meiosis-I). The primary
oocytes mature upon exposure to the hormone
progesterone and reach to the metaphase of the
second meiotic division (Fig. 22.7). The second
Szabad, Biology booklet 22. 3
meiotic division is completed following fertilization
and embryogenesis begins soon with the cleavage
divisions mentioned earlier. When a small sample
taken from a meiosis-II metaphase arrested secondary
oocyte is injected into a meiosis-I G2 phase primary
oocyte, the G2-arrested cell will mature without
progesterone and reach to metaphase of meiosis-II
(Fig. 22.7). Apparently, the G2-arrested Xenopus
primary oocytes are convenient tools to assay MPF:
cytoplasm of basically any eukaryotic cell with MPF
inside will substitute Xenopus MPF and induce
maturation.
In conclusion, the successive steps and direction of
cell cycle progression are assured by cytoplasmic
(MPF, MDF, MPF) as well as chromosome-
associated (re-replication block) factors.
The chromosome cycle
The chromosome cycle includes DNA replication,
the formation of sister chromatids and segregation of
the chromosomes into the daughter cells during
mitosis. (Several features of replication and cell
divisions are discussed in chapters 4 and 5.) DNA
replication proceeds during the S phase of the cell
cycle. DNA replication is semiconservative and bi-
directional. Its speed is 500 and 50 nucleotides/sec in
pro- and eukaryotes, respectively. In prokaryotes it
begins at a single site (in the so-called replication
origo), near the site where the chromosome is
attached to the cell membrane. (See chapter 5.) In
eukaryotes replication begins at several sites during
the onset of the S phase. DNA of the open
chromatin replicates first, as in the genes with house
keeping functions, where DNA polymerase finds
room for action. The highly condensed hetero-
chromatin - including the Barr body chromatin -
replicates last towards the end of the S phase.
Replication of an immunoglobulin-coding gene
complex (composed from about 3x105 base pairs)
begins at several sites inside the complex in those

cells where the gene is expressed. In cells, on the
other hand, where the gene complex is not expressed
the same gene replicates towards the end of the S
phase and its replication starts from a single site
outside the gene complex. One thing is certain: the
chromosomal DNA replicates once and only once
during S phase of the cell cycle. Upon completion of
replication, cells enter G2 to make preparation for
mitosis. Chromatin packaging is the most important
event of G2.
Fig. 22.7. (A) Steps of Xenopus oocyte development
with the arrest points. (B) If, the sample contains
MPF, the meiosis-I G2 arrested cell matures to
meiosis-II metaphase secondary oocyte. (C) If,
however, there is no MPF in the injected sample, the
Xenopus primary oocyte remains in meiosis-I G2.
Are there special sites where replication begins? In
a set of experiments, DNA polymerase was added to
yeast DNA. Although the DNA polymerase
complexes associated with the DNA, replication did
not commence since there were no nucleotide
triphosphates provided. DNase enzyme was added
next to digest DNA sections that were not protected
by the DNA polymerase complexes. The enzymes
were subsequently inactivated and the remaining
DNA fragments were isolated and sequenced. It has
turned out that there are 11 base pair long so-called
autonomously replicating sequences (ARS) in the
yeast DNA where DNA polymerase molecules bind
and begin replication. The ARS consensus sequence
is this: 5 A(T)TTTATA(G)TTTA(T)3 . ARS, like
the TATA box, is rich in A=T base pairs and allows
Szabad, Biology booklet 22. 4
easy separation of the two DNA strands. Several
ARS are frequently present over some 100 bp
stretches of the DNA and recognized by the so-called
replication initiating proteins.
Telomere shortening and telomerase function
In eukaryotes DNA in the nuclear chromosomes is
not ring but rather rod shaped. Knowing molecular
features of replication, it is to be expected that the
lagging strand will become shorter and shorter along
progression of the successive rounds of replications.
(See also chapter 5.) Shortening chromosome termini
leads to loss of essential genes and consequently
death of the cell. Function of the telomeres is to
prevent chromosome shortening (Fig. 22.8). Short
DNA sequences are tandomly repeated in the
telomeres (GGGTTA in humans). Telomere
shortening is avoided through activity of the so-
called telomerase enzyme. Telomerase binds to the
parental DNA strand and extends in the 5 3
direction (Fig. 22.8). In absence of a template strand
telomerase uses, as template, its own RNA to add
single stranded blocks of the repetitive DNA (Fig.
22.8). The RNA is a component of telomerase. As
consequence of telomerase activity, the parental
strand becomes long enough for synthesis, by DNA
polymerase, of the complementary DNA strand (Fig.
22.8).

Fig. 22.8. The mechanism of telomerase action.
There are no telomerase molecules in the so-called
teminally differentiated somatic cells and thus
telomeres become shorter and shorter along
progression of the successive cell cycles. The
phenomenon called telomere shortening limits the
number of cell divisions and is an important factor of
aging. Telomerase is expressed in the germ line cells
and also in the so-called stem cells which replace the
lost somatic cells. The stem cells thus ensure
unlimited numbers of cell divisions. Quite
unfortunately, the telomerase-coding gene is
expressed in the tumor cells. (See chapter 23.)
Fig. 22.9. Events of the centrosome cycle.
Centrosome and cytoplasm cycles
The centrosome cycle implies formation of
devices in the cells that ensure arrangement of the
chromosomes in the metaphase plate, chromosome
segregation, and separation of the daughter cells.
Szabad, Biology booklet 22. 5
Centrosomes are of basic importance in organizing
the spindle apparatus from mitrotubules (Fig. 22.9;
see also Chapters 2 and 19).
Cells carry out their functions during interphase.
During mitoses cells concentrate on mitotic
functions. Cells that can not afford suspending their
functions do not divide. Heart muscle cells represent
a good example for the non-dividing cell types. Other
cells become polyploid, yet others as the
Drosophila salivary gland cells become ploytenic
each with a giant chromosome. The cytoplasm cycle
means sharing of the mother cell cytoplasm between
the daughter cells such that both daughter cells will
inherit cell components like the centrosome and the
mitochondria.
Genetic dissection of the cell cycle
How could one determine molecular nature of the
cell cycle components, including SPA and MPF? The
solution came largely from two species of yeast and
genetic dissection. Yeast are unicellular eukaryotic
organisms, which allow convenient analysis of the
cell cycle (Fig. 22.10). If cell cycle progression
control factors are proteins, function of their
encoding so-called cell-division-cycle (cdc+) genes
can be eliminated by mutations. However, loss-of-
function mutations in the cdc+ genes are lethal. The
so-called conditional lethal mutations provide an
elegant solution. Conditional lethal mutations allow
normal development under permissive conditions but
bring about death under restrictive conditions. The
yeast cdc mutations may live and proliferate on 30 oC
but die on 37 oC. (See also chapter 6.) It may also be
expected that cell cycle progression will came to a
standstill in the cdc mutants under the restrictive
conditions at the G1/S or at the G2/M check points.
Following random mutagenesis, the cdc mutant yeast
cells that can not progress cycle beyond G1/S or
G2/M, naturally under restrictive conditions, were
isolated. The cdc mutations thus identify genes with
functions in cell cycle progression control. Are there
ways to elucidate molecular functions of the cdc-
identified cdc+ genes?
 Szabad, Biology booklet 22. 6

present in the cytoplasm throughout the cell cycle.

Cdks combine with cyclins and attain kinase
activities (Fig. 22.13). (Kinases transfer phosphate
groups, from ATP, to OH groups onto side chains
of amino acids in protein molecules.) In humans,
SPA is Cdk4 combined with cyclin D (Fig. 22.13).
The Cdk4/cyclin D complexes phosphorylate those
proteins that initiate DNA replication. MPF is a
complex of Cdk2 and cyclin B (Fig. 22.13). Kinase
activities of the Cdk2/cyclin B complexes lead to,
among others, disassembly of the nuclear envelope in
mitotic prophase, or the packaging of chromatin for
chromosome formation. Following cyclin dacay (Fig.
22.12) activities of the ever-present phosphatases
become apparent. Phosphatases remove the
phosphate groups that the kinases put onto the
protein molecules. The appearance and disappearance
of the diferent types of cyclines and their
combination with Cdks provide bases of cell cycle
progression.

Fig. 22.10. Life cycle of the budding yeast

(Saccharomyces cerevisiae) and the fission yeast
(Schizosaccharomyces pombe). While budding yeast
cells live their life in diploid conditions, the fission
yeast cells are haploid.

Let s prepare a culture of cdc mutant yeast cells

(Fig. 22.11). Bring plasmids, through transfection,
into the yeast cells. The plasmids carry different
sections of the yeast genome. Plate the yeast cells
onto food surface in Petri dishes, and transfer the
dishes onto restrictive temperature. Under restrictive
conditions only those cells can divide and form
colonies, which carry in the plasmid the normal cdc+
allele of the cdc mutation (Fig. 22.11). The plasmids
Fig. 22.11. Scheme to isolate cdc mutation-identified
can than be isolated form the yeast cells as well as the
genes.
cdc+ allele-containing yeast genomic sequence from
the plasmids, and eventually molecular function of
the cdc+ gene can be elucidated. Cloning several of
the cdc+ genes clearly showed that the cell-division-
cycle control genes are evolutionary
highly conserved, just like the cell
division machinery.

Cell cycle in light of molecules

It has turned out, following analysis of
cdc+ gene functions, that concentration of
the cdc13+-encoded protein cycles during
the cell cycle (Fig. 22.11): the protein
concentration is very low at the end of the
M phase, increases throughout interphase,
peaks at metaphase and quickly decreases
again. The protein is an example of the
cyclins. The cdc13+-encoded protein is
the main mitotic cyclin, cyclin B (Fig. 22.11), and
not only in yeast, but also in most eukaryotic cells. Fig. 22.12. The change of MPF activity and cyclin
Product of the fission yeast cdc2+ gene (cdc28+ in concentration during successive cell cycles.
budding yeast) encodes a cyclin-dependent-protein-
kinase (= Cdk). The Cdk molecules are continuously
 Szabad, Biology booklet 22. 7

Table 22.1. Division capacities of fibroblast

cells of different origin.
Source of cells Number of cell division
Fetus ~50
Forty year old man ~40
Eighty year old man ~30
Werner syndrome people 0

Cell communication and cell proliferation

Fig. 22.13. The bases of SPA and MPF activities are

cyclic appearance and combination of the cyclins
with Cdks along progression of the cell cycles.

It is understandable that the Cdk/cyclin complexes

play essential functions in tumor formation. Cyclin D
concentration is exceedingly high in some types of
breast cancers. There are a number of gain-of-
function (dominant) mutations known in cyclin genes
that encode for the synthesis of mutant cyclin
molecules. The mutant cyclin molecules activate the
Cdk molecules in unusual stages of the cell cycle and
induce excessive cell proliferation. In about 50% of
the tumors function of the p53 gene is defective. The
normal p53-encoded protein blocks Cdk4 activity. In
absence of p53 protein Cdk4 drives cells to S phase,
uncontrolled cell proliferation and tumor formation.
The failures listed above are examples for defects
that lead to excessive cell proliferation. Naturally,
there are mutations known that eliminate cell cycle Fig. 22.14. Cells in contact with neighboring cells
and cell proliferation. The Werner syndrome people do not divide underlying the importance of cell-to-
are homozygous for a mutation in a DNA helicase cell communication in regulation of cell cycle
gene required for separation of the DNA strands control.
during replication. Werner syndrome people age fast
since their cells seldom if ever divide. While
fibroblast cells isolated form healthy people divide in Cells in our body do not divide randomly. Cells
appropriate cell cultures, fibroblast cells from Werner know about each other and are constantly
syndrome people do not divide (table 22.1). communicating. Apparently there are extracellular
 Szabad, Biology booklet 22. 8

factors that regulate cell cycle. Of the extracellular

factors cell-to-cell communication, hormones and
growth factors are of outstanding importance. The
importance of cell-to-cell communication is nicely
indicated by the observation that cells in cell culture
medium proliferate and spread as long as there is free
surface available. Cell proliferation ceases when cells
cover the entire surface and begin to communicate,
block each other s division through the phenomenon
called contact inhibition (Fig. 22.14). Cell division
will resume only after removal of cells (Fig. 22.14).
Please notice that only those will divide that had lost
the neighboring cells.

SUMMARY
Billions of cells compose body of many of the
multicellular organisms: they are at the right place in
the right time and carry on their characteristic
functions. Billions of cell cycles take place up to
development of the multicellular organisms like
humans. Development is achieved through
appropriate mechanisms that ensure appropriate
control of cell cycle progression. Understanding
molecular nature of cell cycle control provides basis
to deal with tumor formation.

REFERENCES
1. Purves, W.K. et al., Life the Science of Biology,
193-215, 1998.
2. Alberts et al., Essential Cell Biology, 572-592,
1998.
3. Lodish, et al., Molecular Cell Biology, 9-11,
495-531, 2000.