Download ISSN-1916-5382 Title: Factors Regulating Cell Division in eukaryotic

ISSN-1916-5382
Title: Factors Regulating Cell Division in eukaryotic cells
Authors: Jigna G. Tank, Rohan V. Pandya and V. S. Thaker
Affiliation: Centre for Advanced studies in Plant Biotechnology and Genetic Engineering
(CPBGE), Department of Biosciences, Saurashtra University, Rajkot- 360005
Abstract
Cell cycle is a key process in growth and development of living organisms. It is regulated at
each and every step during growth. There are various factors responsible for regulating cell
cycle for better development of an organism. These factors are regulated by biotic and abiotic
environmental conditions. Biotic factors includes cell cycle checkpoints used during each cell
cycle phases, Cyclin dependent kinases (CDKs) enzymes regulating the process of cell
division and Cell cycle inhibitors. Abiotic factors includes in vivo regulators and in vitro
regulators. In vivo regulators are nutrients, environmental factors and hormones. In vivo
regulators includes various chemicals responsible for cell synchronization, natural products
used as antimitotic agents and miscellaneous agents used for cell cycle inhiition
Introduction
Cell cycle is the process of formation of two daughter cells from parent cell. It is a key
process in the formation of all living organisms. it is divided into two phases:
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M phase:
During M phase, cellular contents are partitioned between two daughter cells. It is composed
of two tightly coupled processes: (a) mitosis, in which the cell’s chromosomes are divided
between the daughter cells and (b) cytokinesis, in which the cell’s cytoplasm divides forming
distinct cells.
Interphase:
After M phase, each daughter cell begins interphase of a new cycle. All stages of the
interphase are not morphologically distinguishable. Interphase is formed of three stages G1
phase, S phase, and G2 phase. In G1 phase the cell grows; in S phase the nuclear genetic
information is replicated and in G2 phase further growth of cell occurs in preparation for
division. Each phase of cell cycle has a distinct set of specialized biochemical processes that
prepare the cell for initiation of cell division.
a) G1 phase: G1 phase starts with the end of M phase and ends with the beginning of DNA
synthesis, indicating gap or growth. This phase is involved in synthesis of various enzymes
that are required in S phase. The biosynthetic activities of the cell are resumed at a high rate
during this phase, which has been considerably slowed down during M phase. Duration of
G1 phase is highly variable.(Smith et al, 1973 )
b) S phase: it starts with DNA synthesis and completes when all of the chromosomes have
been replicated. Although the Ploidy of the cell remains the same, the amount of DNA is
double during this phase. Each chromosome has two sister chromatids. Except for the high
histone production rate of protein synthesis and RNA transcription are very low in S phase.
The duration of S phase is relatively constant. (Cameron et al, 1963 ).
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c) G2 phase: After DNA replication, cell enters G2 phase. During this phase Protein
synthesis occurs which involves production of microtubules, in turn required for mitosis.
Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis. It
lasts until the cell enters the next round of mitosis.
d) G0 phase: cells that have temporarily stopped dividing are said to have entered a state of
quiescence called G0 phase. Non-proliferative cells in multicellular eukaryotes generally
enter the quiescent state from G1 and may remain quiescent for long period of time. This is
very common for cells that are fully differentiated. Some non-proliferative cells remain
quiescent indefinitely for example, neurons. While others such as epithelial cells, continue to
divide throughout an organism’s life (Wikipedia free encyclopedia, 2007). Plant cells can be
induced to begin dividing again under specific circumstances. They have ability to
dedifferentiate and acquire pluripotency, a phenomenon known as totipotency, which is not
found in animals.
New organs in plants such as roots, stem, leaves and flowers originate from life-long iterative
cell divisions followed by cell growth and differentiation. Such cell division occurs at
specialized zones known as meristems. The shoot and floral meristem forms leaves and
flowers whereas, the root meristems causes the growth of roots. The cells at the meristem are
pluripotent, so that their progeny can acquire a spectrum of developmental fates. For
example, initially the shoot apical meristem produces leaves under the right developmental or
environmental condition; it gets converted into a floral meristem that produces flowers.
The study of genetic, biochemical and molecular lines during the last decade has shown that
four phases of cell division are regulated by many factors. Cell cycle regulators regulate all
the phases of the cell cycle by regulatory pathways that communicates with environmental
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constraints such as nutrient availability, signals such as growth factors or hormones and
developmental cues which controls the cell division and determines when and in which cell,
division will occur.(Doerner, 1994, Inze and De Veylder, 2006).
Factors regulating cell cycle:Factors regulating cell cycle are divided into two main types:
I.
Abiotic factors
II.
Biotic factors
Abiotic factors:Abiotic factors are divided into two types:
I.
In vivo regulators
Nutrients and Environmental factors:
Cell cycle is regulated by the restriction point which is present in late G1 phase. It controls
progression of cell cycle from G1 to S transition. Once cell have passed restriction point, it is
allowed to enter S phase and thus it undergoes one cell division cycle. However, passage
through the restriction point is a highly regulated process. It is controlled by external signals,
such as availability of nutrients and environmental conditions. Restriction point is a decision
point which determines whether sufficient nutrients are available with the cell to progress for
the cell division. Restriction point also controls the size of the cell. It is the point at which
cell growth is coordinated with DNA replication and cell division. In order to maintain
constant size, the small daughter cell must grow up to certain size before they divide again.
The cell size must be monitored in order to coordinate cell growth with other cell cycle
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events. The cell growth is regulated by a control mechanism that requires each cell to reach a
minimum size before it can pass restriction point. Cell cycle in plants, is regulated by the
extracellular growth factors that signal cell proliferation. In the presence of appropriate
growth factors, cell passes the restriction point and enter S phase. Once it has passed through
restriction point the cell is allowed to proceed through S phase and rest of the cell cycle, even
in absence of further growth factor stimulation. If appropriate growth factors are not
available in G1phase then progression of the cell cycle is stopped at the restriction point. Cell
cycle in plants, is also regulated at G2 to M transition where cell size and nutrient availability
are monitored(Geoffrey, 1997).
Hormones
Hormones play a necessary role in cell proliferation. Cytokinins and auxins the hormones
linked with cell proliferation. They play a crucial role in maintaining undifferentiated cells
for proliferation during in vitro culture. In 1994 Jacqmard et al. suggested that cytokinins
have effects on the G1/S and G2/M transitions, as well as on progression through S phase.
Naturally occurring cytokinins are involved in the control of a wide range of biological
activities. The hormone cytokinin induces the synthesis of specific cell division proteins
which are required for progression of cell cycle. (Genschik et al., 1998). These proteins must
be synthesized according to a genetically determined program for progress of cell cycle.
(Cleary et al., 2002). Cytokinin is also involved in the activation of CDKs at the G2/M
boundary either by direct activation of a phosphatase or by downregulation of the WEE1
kinase. Cell division is inhibited by abscisic acid. Abscisic acid induces the expression of the
CDK inhibitor KRP1/ICK1, resulting in a decrease in the kinase activity associated with
CDKA (Wang et al 1998). Jasmonic acid application during G1 prevents DNA synthesis,
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whereas jasmonic acid application during early S phase has only a minor effect on DNA
synthesis but it inhibits M phase progression (Swiatek et al., 2002). Ethylene treatment of
cell suspensions provoked cell death at the G2/M boundary (Herbert et al., 2001).
II.
in vitro regulators
Potent and selective chemicals mediate inhibition of the cell cycle and are capable of
arresting cell in a particular phase of cell cycle. These chemicals are capable of arresting cell
cycle via specific interaction with a variety of intracellular protein targets and by genetic
restrictions (Crews and Shotwell, 2003). Chemicals involved in cell synchronization are
grouped as below:
A. Synthetic (Artificial) Products
a) Alkylating Agents
b) Antimetabolites
c) Natural products
d) Miscellaneous agents
Synthetic products:These are the products synthesized artificially from various chemicals
A)
Alkylating agents:
These agents are capable of introducing alkyl group into biologically active chemical
molecules. They cause miscoding, destruction of guanine, and disruption of nucleic acid
function. The alkylating occurs due to some chemical groups like bis (2-chloroethyl) group
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which have a property to form covalent bond with suitable nucleophilic substance in the cell.
The main action of alkyalating agents occurs during replication. They affect S-phase leading
to blockage in G2-phase and subsequent apoptotic cell death (Goyal et al, 2004). Alkylating
agents such as cyclophosphamide, chlorampucil, and melphalan are used to block cell cycle
in G2-phase.
B)
Antimetabolites :
Antimetabolites affect synthesis of purines, pyrimidines ,and thereby affect nucleosides.
They block cell cycle in S-phase. Antimetabolites prevent the biosynthesis or utilization of
normal cellular metabolites. They combine with the same enzymes as physiologically
ocuring cellular metabolites but they prevent the metabolic process (Singh and Kapoor,
1996).
Antimetabolites include folic acid analogues, purine analogues and pyrimidine
analogues (Goyal et al, 2004).
Folic acid is essential for synthesis of purine nucleotides and thymidylate, which in turn are
essential for DNA synthesis and cell division (H.P. Rang et al, 2005).folic acid is also
required for interconversion of aminoacids like serine to glysine, histidine to glutamic acid
etc (R.K. Goyal et al, 2004). The main action of the folate antagonists is to interfere with
thymidylate synthesis. Folates consist of three elements: a pteridine ring,p-aminobenzoic acid
and glutamic acid. Folates are actively taken up into the cells where they are converted into
polyglutamates. In order to act as coenzymes, folates must be reduced to tetrahydrofolate
(FH4 ). This reaction is catalysed by tetrahydrofolate reductase enzyme. FH4 functions as a
cofactor in the transfer of one- carbon units, a process which is essential for the methylation
of the uracil in 2-deoxyuridylate (DUMP) to form thymidylate (DTMP) and hence for the
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synthesis of DNA and for the de novo synthesis of purines.During the formation of DTMP
from DUMP, FH4 is converted back to FH2. The folic acid analogues, such as methotrexate
has a high affinity for dihydrofolate reductase than FH2; it thus inhibits the enzyme and
depletes intracellular FH4. The binding of methotrexate to dihydrofolate reductase involves
addition of hydrogen bond or ionic bond not present when FH2 binds (H.P. Rang et al,
2005).
Purine analogues are a group of relatively specific CDK inhibitors that are useful for basic
cell biology and are lead compounds for antiproliferative therapeutics. Purine analogues are
substance that affect the metabolism of the biochemical pathway involved in the synthesis of
purines and pyrimidines (R.K.Goyal et al, 2004).
Natural products :-
These are products extracted from plants, which are used as antimitotic agents. It involves
substances such as Colchicine, which is a highly poisonous alkaloid extracted from plants of
the genus Colchicum, Vinca alkaloids extracted from plant Vinca rosea etc. (Shown in
Table: 1)
Miscellaneous agents:Miscellaneous agents such as hydroxyurea, procarbazine, mimosine and nocodazole are
involved in cell cycle inhibition. Mode of action of chemical cell cycle regulators are shown
in the following table:
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Inhibitors of cell
cycle
Cyclophosphamide
Chlorampucil
Melphalan
Methotrexate
Fluorouracil
Cytarabine
Mercaptopurine
Mode of action
Alkylating agents
It forms covalent bond
with suitable
nucleophilic substance
in the cell.
It forms covalent bond
with suitable
nucleophilic substance
in the cell.
It forms covalent bond
with suitable
nucleophilic substance
in the cell.
Antimetabolites
As folic acid is
required in purines and
pyrimidines synthesis.it
binds to dihydrofolate
reductase.as a result
folic acid cannot be
converted to folinic
acid.
It inhibits thymylate
synthatase enzyme
which is required for
thyminemonophosphat
e formation.
It causes
phosphorylation
reaction to form
arabinosecystidine
monophosphate which
get incorporatedinto
both RNA & DNA.
It causes accumulation
of 6 thioguanosine 51
phosphate & 6
thioinosine 51
phosphate which
incorporate into
cellular DNA.
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Cell cycle phase
arrested
References
G2 phase
Rang et al.
2003
G2 phase
Rang et al.
2003
G2 phase
Rang et al.
2003
S phase
Kaufman et
al. 1993
S phase
Pressacco et
al. 1996
S phase
Zarilli et al.
1999
S phase
Rang et al.
2003
Azathioprine
Amphidicolin
Thioguanine
Lavendustin A
Radicicol
Lactocystin
PS-341
Epoxomicin
Aclacinomycin A
It causes accumulation
of 6 thioguanosine 51
phosphate & 6
thioinosine 51
phosphate which
incorporate into
cellular DNA.
Inhibitor of DNA
polymerase a and d in
eukaryotic cells
It causes accumulation
of 6 thioguanosine 51
phosphate & 6
thioinosine 51
phosphate which
incorporate into
cellular DNA.
It is an inhibitor of
several protein tyrosine
kinases including
epidermal growth
factor receptor (EGFR)
PTK and the nonreceptor PTK, SYK.it
is moderately effective
inhibitor of tubulin
polymerisation
It is an inhibitor of
receptor tyrosine kinase
p60v-src
It covalentely binds to
the N-terminal
threonine of the 20s
proteasome subunit
X/MB1, which is
required for proteolysis
It is a specific inhibitor
of the proteosome
a,b- epoxy ketone class
of proteosome
inhibitors.
It is non-peptide
inhibitor of the
proteosome, showing
selectivity for the
chymotrypsin like and
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S phase
Rang et al.
2003
early S phase
Borner et
al. 1995
S phase
Rang, et al.
2003
G1 and G2 phase
Crews and
Shotwell,
2003
G1 and G2 phase
Soga et al.
1998.
G1 phase
Crews and
Shotwell,
2003.
G1 phase
Crews and
Shotwell, 2003.
Crews and
Shotwell, 2003.
G1 phase
G1 phase
Crews and
Shotwell,
2003.
Trichostatin
Trapoxin
Vinblastine
Vincristine
Colchicine
Pacilitaxel
Epothilonea/B
Discodermolide
PGPH activities of
proteosome.
It is an inhibitor of
histone deacetylase
It is a potent inhibitor
of histone deacetylase
Natural products
It binds to protein
tubulin & inhibits
polymerisation of
microtubules.thus,
prevents spindle
formation & blockage
of mitosis at
metaphase.
It binds to protein
tubulin & inhibits
polymerisation of
microtubules.thus,
prevents spindle
formation & blockage
of mitosis at
metaphase.
It depolymerises
cytosolic microtubules
at higher
concentrations, while at
lower concentrations it
inhibits such
depolymerization
It induces cell cycle
arrest via the
stabilization of tubulin
in an apparently
manner
It induces cell cycle
arrest via the
stabilization of tubulin
in an apparently
manner
It induces cell cycle
arrest via the
stabilization of tubulin
in an apparently
11
G1 and G2 phase
G1 and G2 phase
Gray and
Ekstrom
1998
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
Metaphase
Chan et al.
1998
Metaphase
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
manner
Latrunculin A/B
Jasplakinolide
Mycalolide B
Dolastatin 11
Monastrol
Camptothecin
Hydroxyurea
Procarbazine
L-mimosine
Nocodazole
It binds G-actin and
subsequently
depolymerize actin
filaments
It binds G-actin and
subsequently
depolymerize actin
filaments
It binds G-actin and
subsequently
depolymerize actin
filaments
It inhibits cell cycle by
binding actin, resulting
in reorganization of the
actin filament network
It binds specifically to
the mitotic kinesin Eg5
implicated in spindle
bipolarity
Miscellaneous agents
It inhibits DNA
topoisomerase I
It inhibits
ribonucleoside
diphosphate reductase
that catalyses
conversion of RNA to
DNA.
It inhibits protein and
nucleic acid synthesis
and supress mitosis
It is an effective
inhibitor of DNA
replication. It acts by
preventing formation of
replicative forks
Promotes tubulin
depolymerization
12
Metaphase
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
G2/M phase
Crews and
Shotwell,
2003.
Metaphase
Crews and
Shotwell,
2003.
G2/M phase
G1 phase
Leyden et
al. 2000. ,
G1 phase
Rang, et al.
2003
G1 phase
Lin et al.
1996.
Metaphase
Bunz et al.
1998.
Biotic factors
i. Checkpoints
ii. CDKs
iii. CDK inhibitors
Checkpoints
Cycle checkpoints are used by the cell to regulate cell proliferation. There are two main
checkpoints viz, G1/S checkpoint and the G2/M checkpoint. G1/S Cell is a rate-limiting step
in cell cycle and is also known as restriction point(Robbins and cotran et al, 2004).various
checkpoints work together and would not allow the replication of damaged chromosomes.
This checkpoints will not allow incomplete chromosomes to pass to the new daughter
cells.the G2 checkpoint prevents mitosis until DNA replication is complited.it senses the
unreplicated DNA and then generates a signal which prevents the initiation of M phase. Cell
remains in G2 phase till the DNA is completely replicated. After DNA replication cell is
allowed to enter mitosis and the chromosome are distributed between the two daughter cells.
Cell cycle in G2 check points is also arrested due to DNA damage. This arrest gives time to
DNA to get repaired. DNA damage also arrests the cell cycle in G1. The G1 arrest allow
DNA to get repaired before it enters S phase. If the cell enters S phase without DNA repair
than the damaged DNA would be replicated. One of the important checkpoint that maintains
the integrity of genome in two daughter cells is present at the end of mitosis. It monitors the
alignment of chromosomes on the mitotic spindle. This ensures the accurate distribution of
complete set of chromosomes to the two daughter cells (Geoffrey, 1997).
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Cyclin dependent kinase
The principle regulators of eukaryotic cell cycle at molecular level are the cyclin dependent
kinase (CDK) and cyclins which are conserved in plants (Shaul et al. 1996).cyclin dependent
kinase govern the cell cycle. Different CDK-cyclin complexes phophorylate a plethora of
substrates at the key G1-to-S and G2-to-M transition points, triggering the onset of DNA
replication and mitosis.
The catalytic CDK subunit is responsible for recognizing the target motif (serine or threonine
followed by a proline) present in substrate proteins, whereas exchangeable regulatory
cyclins, play a role in discriminating distinct protein substrates (Inze and De veylder, 2006).
To ensure that the reactions are controlled by the engine are carried out to completion with
sufficient accuracy and in proper order, its activity is feedback regulated at checkpoints
(Hartwell and Weinert, 1989). At checkpoints its kinase activity becomes limiting for further
progress until the feedback control network signals the completion of the dependent
reactions, which then activates the kinase for passage through to the next checkpoint
(Doerner, 1994). Cell cycle checkpoints are used by the cell to monitor and regulate the
progress of cell cycle (Stephen, 1996). Two main checkpoints exist: The G1/S checkpoint
and G2/M checkpoint. G1/S transition is the rate limiting step in the cell cycle and is also
known as restriction point (Robbin and Cotran et al. 2004).
Upon receiving a promitotic extracellular signal, G1 cyclin-CDK complexes become active
to prepare cell for S phase promoting the expression of transcription factors that in turn
promote the expression of S cyclins (Cs) and of enzymes required for DNA replication. The
G1 cyclin-CDK complexes also promote the degradation of molecules that function as S
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phase inhibitors by targeting them for ubiquitination. Once, a protein has been ubiquitinated,
it is targeted for proteolytic degradation by the proteosome. Proteolysis ensures that the cell
cycle moves unidirectionally by trigerring the rapid proteolysis of target proteins, thus
providing an irreversible mechanism that drives the cell cycle forward. Ubiquitin-proteosome
system uses the highly conserved polypeptide ubiquitinin as a tag to mark target proteins for
degradation by the 26S proteosome. Ubiquitination involves the generation of poly ubiquitin
chains on target proteins through the combined action of ubiquitin carrying enzymes (E2s)
and ubiquitin protein ligase (E3s) that bring target proteins and E2s together.
Plants cyclins A and B type cyclins contain destruction box (D- box) sequences that mediate
protein degradation (Genschik et al. 1998, Renaudin et al. 1996).proteins that are degraded
through the proteosome frequently require prior phosphorylation. Proteolysis of target
proteins seems to be proceeded by CDK-dependent phosphorylation. CDKs are
phosphorylated on both an N-terminal Tyr and Thr residue. Tyr phosphorylation is catalysed
by the WEE1 kinase and both Tyr and Thr phosphorylation is counteracted by the dual
specificity phophatase CDc25. Plants possess a WEE1 kinase that is involved in the
inhibitory phosphorylation of CDKs (Sorell et al. 1999,Vandepoele et al. 2002, Sun et al.
1999). Biochemical and genetic evidence suggest that higher plants have a phosphatase that
can activate CDK/cyclin complexes (Wein et al, 2005). Active S cyclin- CDK complexes
phosphorylate proteins that make the pre- replication complexes assembled during G1 phase
on DNA replication origins. The phosphorylation serves two purposes: to activate each
already assembled pre-replication complex, and to prevent new complexes from forming.
This ensures that every portion of cells genome will be replicated only once. Mitotic cyclinCDK complexes, which are synthesized but inactivated during S and G2 phase, promote the
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initiation of mitosis by stimulating downstream proteins that are involved in chromosome
condensation and mitotic spindle assembly. A critical complex activated during this process
is a ubiquitin ligase known as the anaphase promoting complex (APC), which promotes
degradation of structural proteins associated with the chromosomal kinetochore. APC also
targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can
proceed.
Cell cycle inhibitors
Cell proliferation is also regulated by cell cycle inhibitors. During cell division activity of
cyclin dependent kinase is controlled by cdk inhibitors ICK (interactor/inhibitor of CDKs)
and KRPs (Kip-related proteins). They bind with specific CDK/cyclin complex and help in
controlling cdks activity (Tank et al. 2011). During cell division certain specific protein are
synthesized in cell which interact with CDKs to terminate their function. CDK activity can
be terminated by binding, phosphorylation or dephosphorylation of these proteins (Morgan,
1997).
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