Cellular Biology

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Cellular Biology
School of Life Sciences Shaanxi Normal University
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CHAPTER 9 CELL PROLIFERATION AND REGULATION
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The cell cycle can be described as 4 phases
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I. Basic Concepts What is cell cycle A
cell cycle means a cell proliferation procedures
from the end of division to next end of division.
The cell cycle can be described as 4 phases ? G1
phase (gap1phase) means the gap time from the
finished time point of mitosis to the beginning
time point of DNA replication. ? S phase
(synthesis phase) means the time when DNA is
synthesized. H3-TDR can be inserted into new
synthesized DNA in this phase only. ? G2 phase
(gap2 phase) means the gap time from the finished
time point of DNA synthesis to the beginning time
point of mitosis. ? M phase or D phase (mitosis
or division) means the time from the beginning to
ending of mitosis.
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The definition of cell cycle time
Percentage labeled mitoses (PLM) is a regular
method for that. Label cells with pulse labeling
method. Take cell samples at different time
points. Display the labeled cells by
autoradiography. Obtain the percentage of the
labeled cells that proliferated. The terms for
that are as the follows TG1 Consisted time of
G1. TG2 Consisted time of G2. TS Consisted time
of S. TM Consisted time of M. TC Consisted time
of a cell cycle. PLM Percentage of labeled
mitoses. TDR Thymidine. 3H or 14C is used to
label TDR usually. All S cells were labeled by
3H S cells became M cells through G2 phase
(The PLM is 0 in this time) The M cells
appeared, the gap time between PLM 0 and PLM gt
0 is TG2 S cells turned to M cells, and PLM
increased The peak means the late S cells
turned to G1 cells through M phase (PLM 100)
The gap time from M cell appeared to PLM
100 is TM PLM decreased (The time from PLM
appeared to decreased is TS) The time from
PLM appeared to next PLM appeared is TC TC
TG1TSTG2TM Obtain TG1 from this formula
calculation.
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Relationship between the time of each phase and
PLM
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Synchronization Synchronization means
all cells growing in a system are proliferating
in same phase steps of cell cycle. The
synchronization can be formed naturally or
artificially. Natural synchronization
(No presentation, see your text book) Regulated
(Artificial) synchronization 1.Selective
synchronization (1) Mitosis selection When
the cultured cells grow to a logarithmic
proliferation the mitosis cells are separated
from dish bottom because the mitosis cell can not
attach to the dish surface. If you shake the dish
gently, M phase cells will be separated from dish
surface. Collect the cell suspension, spin down
and resuspend the cells with fresh medium again,
continue to culture them. Repeat the steps above,
you will get M phase cells. This method is
simple, easy, and the cells kept no damaged. But
you can not get M cells with 100 ratio. (2)
Centrifuging isolation The cells in different
phases have different sedimentation coefficients.
So, we can isolate each phase cells by centrifuge
method. But the synchronized rate is not so good
if use this method.
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• 2.Regulated synchronization
• Blocking DNA synthesis Use DNA synthesis
  inhibiting reagent to block DNA synthesis
  reversely, regulate all of cultured cells to be
  stopped at S phase or the G ending and S
  beginning. The reagents include
  5-Fluoro-2'-deoxyuridine (FUDR), thymine
  deoxy-ribonucleoside (TDR), ADR, and GDR.
• FUDR and TDR are better than others.
  For example, add excessive TDR into
  logarithmically growing cell culture (Hela
  2mol/L CHO 7.5mol/L), check cell growth each
  day to identify stopped culture growth. Wash
  cells and add fresh medium again and continue to
  the culture. When the time of H3 releasing is
  longer than TS, add excessive TDR again. All
  cells will be stopped at G1/S.
• The advantage of this method is
  very high synchronization rate (almost 100). The
  disadvantage of the method is that the size of
  some cells become bigger.
• (2) Blocking mitosis metaphase Damage
  micro-tubes and stop cell proliferation at
  mitosis metaphase. The reagents include
  colchicine and colchicinamide. Colchicinamide is
  better than colchicine because of its lower
  toxicity.
• The growth of cells by this method
  is good. But it is difficult for the cells to be
  cultured reversely.

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• II. mitosis
• The types of cell division
• Amitosis Amitosis is also called as direct
  division. For amitosis, the nucleus become longer
  and a constriction is formed at middle of the
  long nucleus, then, the nucleus and plasma is
  separated into two new cells. No spindle is
  formed and no chromosome change can be found in
  amitosis. Amitosis can be observed in prokaryotic
  cells and some special eukaryotic cells, such as
  the cells of fetal membrane, muscle cells, and
  others.
• Mitosis Mitosis is also called as indirect
  division. The spindle and chromosome change can
  be observed in mitosis. Generated chromosomes can
  be separated into two new cells equally. Mitosis
  is the popular division style in advanced animal
  and plant cells.
• Meiosis In meiosis, the chromosomes are once
  replicated, but the cells twice proliferated.
  Meiosis is the division style for the germs of
  advanced animals and plants.
• Mitosis
• For the convenience to describe, we divide
  mitosis as 6 stages interphase, prophase,
  premetaphase, metaphase, anaphase, and telophase.
  The interphase include G1, S, and G2 phases in
  that the DNA replication is prepared. I introduce
  other phases involved with mitosis to you as the
  follows

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Prophase In prophase, the following events
happen ? chromatin is condensed ? set up polar
sites and start to form spindle ? nucleolus is
disassembled ? Nuclear membrane (envelope)
disappeared. In prophase, the chromatin
became short and visible under microscope. Each
chromosome include two chromatids. Two
centrioles have already formed in S phase, and
they move to polar sites in prophase. The spindle
microtubes will be formed between the both
centrioles. Spindle is formed, and nuclear
membrane disassembled. The spindle
microtubes include follows ? Kinetochore
mt. ? Astral mt. ? Polar mt or
overlap mt.   The spindle microtubes play
roles to link polar bodies and separate
chromosomes and division bodies. There two types
of motor protein are involved in these changes
dynein and kinesin.
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Both centrioles are moving to polar sites in
prophase
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Premetaphase Premetaphase means the
stage from the membrane disassembly to
chromosomes were managed to equatorial plane.
Spindle microtubes extend into center and combine
to kinetochore.
Left Premetaphase Right Metaphase (From
http//www.wadsworth.org)
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Metaphase Metaphase means the stage from
the chromosomes managed on equatorial plane to
the paired chromatids separated to polar sites.
Left Metaphase Right The microtubes linked to
chromosomes (http//www.wadsworth.org)
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Anaphase In anaphase, paired chromotids
are separated and move to polar sites.
The paired chromotids are separated in anaphase
(http//www.wadsworth.org)
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Anaphase can be described as two stages ?
Anaphase A means the stage when the chromosomes
are moving to the centrosomes (polar sites). ?
Anaphase B means the stage when the distance
between both polar sites is becoming longer.
The changes in both stages above are carried
out by the cooperation of microtubes and motor
molecules. The anaphases A and B were
identified out by using taxol (a reagent) or
others. Taxol can inhibit anaphase A. There is no
anaphase B in plant cells.
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Chromosomes are separating off in anaphase A, and
the both polar sites are moving away each other
in anaphase B
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Chromosomes are separated away by the cooperation
of motor protein and microtubes system
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Telophase Telophase means the stage
from the time point when the new chromosomes
moved to polar sites to the time point when two
new cells have been formed.
Telophase includes new nuclei generation stage
and cell plasma division (cytokinesis) stage.
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New nuclei generation New nuclei
generation takes an opposite way to the one taken
in prophase Chromosome chromatin
appearance of nucleolus envelope
formation. Nucleolus is formed by the
nucleolus organization regions (NORs) of
chromosomes. Plasma division (cytokinesis)
For the eggs of insects, nucleus can be
cleaved many times without cytokinesis.
The cytokinesis of animal cells is started by
forming a contraction ring on plasma membrane. If
the cells are treated by cytochalasin, or
antibodies against myosin or actin, the
contraction ring will not be formed. This
indicates that the contraction ring acts in a
mechanism like muscle contraction.
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The plasma contraction ring of animal cell
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The plasma cleavage of plant cell is
different from animal cell. The microtubes
located in polar sites will disappeared in
anaphase or telophase. But the microtubes located
in center will be remained and generated. So, the
phragmoplast will be formed. The vesicles from
Golgi body will be transported into the
phragmoplast to form the cell plate and the
filament between cells. The vesicles from Golgi
bodies are fused to cell plate to form cell wall,
then, the cell will be separated as two new cells.
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The generation of phragmoplast of plant cell
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• III. Meiosis
• During meiosis, the cell is cleaved
  twice, but the DNA is replicated once only. So,
  the number of chromosome for new generated cell
  is decreased to 23 for each cell (human germ
  cell). So, we say that human germ cells are
  haploid cells (n 23). They will return back to
  diploid (2n 46) by the fertilization with 23
  chromosomes from father and other 23 chromosomes
  from mother. There is an exchange between
  homologous chromosomes by that the genetic
  information for new generation will be
  variegated, the evolution development for species
  will be enhanced, and the number of chromosome
  for cell will be kept no changed consistently.
• Meiosis can be sorted as 3 major
  types
• Gametic meiosis (terminal meiosis). The meiosis
  is combined with germ cell development together.
  For example, one spermatocyte can be cleaved as
  four spermatids through meiosis. The spermatid
  will be developed as sperm. One ovocyte (oocyte)
  can be cleaved as one ovum and 2 or 3 polar
  bodies.
• Sporic meiosis (intermediate meiosis). Sporic
  meiosis is the type of meiosis for plants. Sporic
  meiosis and germ cell development are different
  procedures and not combined together. The
  gametocytes (sporocysts) will be developed as
  haploid microspores and macrospores in sporic
  meiosis. Microspore will be developed as
  microgamete (male gamete), and macrospore will be
  developed as macrogamete (female gamete).

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(3) Zygotic meiosis (initial meiosis). Zygotic
meiosis is the meiosis type for fungi and some
bacteria. The meiosis happens after the
generation of zygote and will form the haploid
sporozoite. Somatic meiosis can be
found in some living things, such as mosquito
larva. Meiosis is composed of both
continued cleavages (Meiosis I and II).
Homologous chromosomes will be separated away in
meiosis I usually. So, we can also call it as
heterotypic division or reductional division.
Paired chromosomes will be separated away in
meiosis II like mitosis. We can also call it as
homotypic division or equational division.
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(No Transcript)
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Model of meiosis
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Like mitosis, the meiosis can be divided as
several stages for the convenience to describe
it. Premeiotic interphase (premeiosis)
Premeiosis includes G1, S and G2 phases. Mitosis
turns to meiosis in G2 phase. Meiosis 1.
Meiosis I Premeiosis I The main steps of
meiosis are carried out in premeiosis I. For the
convenience of statement, we can divide the
premeiosis as 5 stages as the follows (1)
Leptotene Chromosomes appear as filaments with
beaded chromomeres. The chromosomes have
replicated at this time, but the paired
chromatids can not be observed under microscope
yet. This stage is also called as synizesis
because the chromatids are overlapped together.
For some species, the chromatid filament is
linked to the nuclear membrane with one terminal,
and another terminal extends into nuclear plasma.
So, some people call it as bouquet stage. (2)
Zygotene The homologous chromosomes will be
paired in this stage, that is called synapsis.
The homologous chromosomes will form the
synaptonemal complex (SC). We can see the paired
chromosomes combined together under optic
microscope and call it as bivalent. Each pair of
chromosomes is replicated and contains 4
chromatids called as tetrad.
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(3) Pachytene This stage can go on for several
days. Chromosomes will become short and combine
closely each other. The homologous chromosomes
unpaired can be exchanged partially each other at
this time. (4) Diplotene The linked homologous
chromosomes of SC start to separate each other,
and keep a junction at chiasma. SC is disappeared
in this stage. The chiasma can move
to the terminal of the bivalent. We call this
movement as terminalization. The
diplotene is much longer in animal germ cells
than in plant germ cells. Human ovocytes have
turned to diplotene in 5 months fetus, but they
will stay in this stage till to be exported (12
50 years old woman). (5) Diakinesis The
bivalents become short, move to the peripheral
area of nucleus, and distribute to every where
in nucleus. So, this stage is the best time to
observe chromosomes in meiosis cells! The
shape of bivalents can be V or O at this time.
Nucleolus will disappear at this time. But
some plants, such as corn, keep nucleolus visible
still at this time.
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Metameiosis I (Metaphase I) Nucleolus
disappeared and nuclear membrane disassembled
indicate that the cell has turned to metameiosis
I. In this stage, chromosomes are arranged on the
equatorial plane. Each bivalent has 4
centromeres, and the centromeres of paired
chromatids are located to the polar side of
spindle. We call this localization as
co-orientation. Anameiosis I The
homologous chromosomes of bivalent are separated
and move to both polar sites. The number of
chromosomes in each polar area is decreased by
half because the separation of homologous
chromosomes. The separation and distribution of
homologous paired chromosomes in both polar sites
is absolutely randomly to make the recombination
of the chromosomes from mother and father, that
is very beneficial to the genome mutation. There
are 23 pairs of chromosomes in a human cell with
223 recombination types. So, excepting
monozygotic twins, it is almost impossible to
have the generations with same genetic
features. Telomeiosis I After
chromosomes move to polar areas, they are
despiralized, and the nuclear membrane and
nucleolus will be assembled again. Meantime, the
plasma will be cleaved.
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Intermeiosis It is a short stage between
meiosis I and meiosis II without DNA
synthesis. 2. Meiosis II It is similar to
mitosis, and can be divided as 4 stages
premeiosis II, metameiosis II, anameiosis II, and
telomeiosis II. By the meiosis, a spermatocyte
can form 4 sperms, and a ovocyte can form 1 ovum
and 2 or 3 polar bodies. Synaptonemal complex
(SC) Synaptonemal complex (SC) is formed
by two homologous chromosomes in the zygotene. SC
is associated with the pairing, exchanging, and
separating of homologous chromosomes. The both
sides of SC are 40nm lateral element, and a 100nm
intermediate space is located in center. A 30nm
central element is located in center of
intermediate space. The 7 10nm SC fibers are
horizontally arranged between lateral element and
central element, that is make SC looked like a
ladder. The spherical recombination
nodules (RNs) can be observed in the SC stained
by phosphotungstic acid. RNs are the sites where
the homologous chromosomes are crossed. Some
enzymes for gene exchange are located on RNs.
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SC fibers
Lateral element
Intermediate space
Lateral element
A SC of an insect
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IV. Regulation of Cell Cycle The background of
research on the regulation Rao and
Johnson (1970?1972?1974) synchronized cultured
Hela cells at different phases, then, mixed them
with M phase cells to induce the cell fusion
intermediated by inactive Sendai virus. They
found that the interphase cells of the fused
cells can form the different shapes of
prematurely condensed chromosome (PCC). They
named this course as premature chromosome
condensation. The PCC of G1 phase is
leptotene because the DNA is not replicated yet.
The PCC of S phase is like powder because
the DNA has been replicated on many sites.
The PCC of G2 phase is diplotene because the DNA
replication has been finished.
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The PCCs with different shapes
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The described above just shows you that
the fused M phase cells of same type of cells can
introduce PCC. Actually, the fusion of different
types of cells can introduce PCC also. For
example, the fused M phase cells of human and
toad can introduce PCC also. The result above
indicates that M phase cells can secret some
factor that can promote the proliferation of
interphase cells. The factor is called as
maturation promoting factor (MPF). In
1960s, Yoshio Masui found the extract from the
matured frog egg can promote the Germinal Vesicle
Breakdown (GVBD) of immature frog egg. Sunkara
injected the extract from different phase Hela
cells into frog egg, they found that the extract
from G1 and S phase cells can not introduce GVBD,
but the extract from G2 and M cells can promote
GVBD. They named this extract as mitosis factor
(MF). The same factors were found in many other
types of cells later on. All of they are called
as MPF.   In 1960s, Leland Hartwell
isolated tens cell division cycle gene (CDC) from
some yeast cells. For example, the cdc28 gene
plays an important role on the G2/S exchange. By
the research on the sensitivity of yeast to
radioactive rays, Hartwell put forward a new
concept, checkpoint, means that a cell cycle will
be stopped when the DNA is damaged.
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Cell cycle of yeast
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Cell cycle of yeast
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In 1970s, Paul Nurse, et al, found many
cell cycle regulatory genes from yeast. For
examples, the mutants of cdc2, cdc25 can not take
mitosis at regulated temperature, the mutant of
wee1 can start division early, and the mutant
cdc25 and wee1 can take division normally. By the
further experiments, they found cdc2 and cdc28
encode a 34KD protein kinase to promote cell
cycle. The genes wee1 and cdc25 can inhibit or
enhance cdc2. That is why the mutant of cdc25 and
wee1 can take normal division. In 1983,
Timothy Hunt found some special protein from the
fertilized egg of sea urchin firstly. The level
of the protein in the each phase of the egg cell
cycle is changed obviously. This protein
synthesis can be started at G1, increased to a
peak at G2/M, and disappear after M phase. He
named the protein as cyclin. Cyclin was lately
found in other animals and yeast. The cyclin
mRNAs from other animals can promote the
maturation (division) of the fertilized frog egg.
Because the great contribution to the
cell biological development, all persons above
became Nobelist later.
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If the expression of Cdc25 is inefficient, the
cell grows long without division If the
expression of Wee1 is inefficient, the cell
starts division early (Cell is small)
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In 1988, M. J. Lohka purified the MPF from
toad, and found that the MPF is composed of 32KD
and 45KD proteins, the complex of the both
proteins can phosphorylate many other proteins.
Paul Nurse(1990)found that the 32KD and 45KD
proteins are the homologous molecules for cdc2
and cyclin B. This finding combines the main
research projects above together. On Oct. 8,
2001, American, Leland Hartwell, British, Paul
Nurse and Timothy Hunt won the Nobel prize
because of their great contribution to the
researches on the cell cycle regulation.
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34KD protein kinase
MPF cdc2 Cyclin B
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Cyclin-dependent kinase (CDK) Cdc2 can
get kinase activity when it combines to cyclin,
so, cdc2 is called as cyclin-dependent kinase
(CDK) or CDK1. Activated CDK can phosphorylate
proteins to take functions. For example, it can
phosphorylate lamina protein to disassemble
nuclear skeleton and envelope. By this
disassembly, the cell cycle can be kept for
continued division. So far, 7 CDKs (CDK1
CDK7) have been found in animals. Each of them
contains a similar kinaselike domain with a
conserved peptide sequence, such as PSTAIRE, that
is the binding site to cyclin. CDK inhibitor
(CDKI) There are CDK inhibitors (CDKI)
in cells that can regulate cell cycle negatively
(inhibiting). Two families of CDKI have been
found so far ? Ink4 (Inhibitor of cdk4), such
as, P16ink4a, P15ink4b, P18ink4c, and P19ink4d.
These Ink4s can inhibit the complex of
cdk4cyclin D1 and cdk6cyclin D1 specifically. ?
Kip (Kinase inhibition protein), such as, P21cip1
(cyclin inhibition protein 1), P27kip1(kinase
inhibition protein 1) and P57kip2 that inhibit
the kinase activity of most of CDKs. P21cip1 can
combine the helper factor of DNA polymerase,
proliferating cell nuclear antigen (PCNA), to
inhibit DNA synthesis.
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CDK and PCNA are inhibited by P21cip1
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Cyclin Cyclin can both activate CDK and
manage what substrate will be phosphorylated
where and when to promote cell cycle. So far,
more than 30 cyclins have been isolated from
yeast and animals. The cyclins in vertebrates
include A1-2, B1-3 , C, D1-3, E1-2, F, G, and H.
All of them can be sorted as 4 types G1, G1/S,
S, and M. Each type of cyclin contains an about
100aa (amino acid) conserved sequence called as
cyclin frame that can intermediate the
combination of cyclin and CDK. Cells will
express G1 phase cyclin D under the growth factor
activation. The cyclin D can combine CDK4 and
CDK6 to phosphorylate the downstream proteins,
such as Rb, and the phosphorylated Rb can release
out the transcription factor, E2F, to promote
many transcriptions of genes, such as, the genes
of cyclin E, cyclin A, and CDK1.
Cyclins
Complex of kinase Vertebrates Vertebrates Yeasts Yeasts
Complex of kinase Cyclin CDK Cyclin CDK
G1-CDK Cln D CDK4, 6 Cln 3 CDK1(cdc28)
G1/S-CDK Cln E CDK2 Cln 1?2 CDK1(cdc28)
S-CDK Cln A CDK2 Clb 5?6 CDK1(cdc28)
M-CDK Cln B CDK1(cdc2) Clb 1-4 CDK1(cdc28)
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The combination of Cyclin D and CDK promotes Rb
to release out the combined transcription factor,
E2F
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In G1/S, cyclin E combines CDK2 to promote
the cell turn to S phase through G1/S restriction
point. If you use antibody against Cyclin E to
the cultured cells, your cells will be stopped at
G1. If you use antibody against Cyclin A, the DNA
synthesis in your cells will be inhibited.
In G2/M, cyclin A and cyclin B can combine CDK1
to phosphorylate substrate protein to cause the
downstream events. For examples, phosphorylation
of histone H1 can cause the condensation of
chromosomes, and phosphorylation of lamina
proteins can cause the disassembly of envelope.
The cyclic changes of cyclin
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In the metaphase with peak of MPF activity, by an
unknown pathway, anaphase promoting complex
(APC), can be activated to combine ubiquitin to
cyclin B, and cause cyclin B degenerated by
proteasome. A cell cycle is finished.
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There is a sequence associated with cyclin
destruction on the N terminal of mitosis phase
cyclin. We call this sequence as destruction box.
At the peak of MPF, the ubiquitin ligase can
promote the combination of ubiquitin and cyclin.
The ubiquitin combined cyclin can be hydrolyzed
by 26S proteasome. G1 phase cyclin can be
hydrolyzed by same way, but there is no
destruction box on it. There is a PEST sequence
on the C terminal of it that is associated with
its destruction. Ubiquitin is composed of
76 amino acids, it is highly conserved, and
exists in all eukaryotic cells. That is why it is
called as Ubiquitin. Ubiquitin functions like a
marker to be destructed. All proteins combined
ubiquitin can be recognized and destructed by
proteasome. It is the popular way by that short
life proteins and abnormal proteins in cells can
be cleaned. In the polyubiquitination,
E1, an ubiquitin-activating enzyme, gets energy
by hydrolyzing ATP to activate ubiquitin, then,
E1 transfers the activated ubiquitin to E2, an
ubiquitin-conjugating enzyme. Finally, E3, an
ubiquitin-ligase, links the ubiquitin to target
protein. There are two types of ubiquitin ligases
at least involved with cell cycle regulation
skp1-cullin-F-box protein (SCF), a complex
composed of 3 proteins, links ubiquitin to G1/S
cyclin and some CKIs, APC will combine the
ubiquitin to M phase cyclin like what is shown by
the fig above.
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The destruction box and cyclin destruction
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DNA replication should be once carried out, and
is once carried out exactly in a cell cycle
DNA replication is started from the origins of
replication that are the autonomously replicating
DNA sequences (ARSs) distributed in chromosome,
and I presented them to you in last chapter. In
the cell cycle, these DNA replication origins are
combined with origin recognition complex (ORC)
that can be bound by other regulatory factors.
Cdc6 is the one of these factors. Another very
important factor is from a special protein family
named minichromosome maintenance protein (MCM).
MCM is the licensing factor to the DNA
replication, and 6 MCMs (MCM2 MCM7) have been
found. If any one of MCMs is absented, the DNA
replication will be stopped. In G1 phase, the
level of cdc6 is increased quickly, and it is
combined to ORC to promote other proteins
including MCM binding to ORC, and form the
pre-replicative complex (pre-RC). MCM is DNA
helicase actually. S phase CDK (S-CDK)
both triggers pre-RC to start DNA replication and
blocks the DNA re-replication because S-CDK can
phosphorylate cdc6 to separate it from ORC.
Phosphorylated cdc6 can be degenerated by the SCF
joined polyubiquitination pathway. S-CDK can also
phosphorylate MCM to inactivate it. Other CDKs
can also block the pre-RC secondary formation. By
all pathways described above, it is ensured that
the DNA replication should be carried out once
only, and is carried out once only in a cell
cycle.
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Each cell cycle triggers DNA replication once
only (from Molecular Biology of the Cell 4th ed.)
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M CDK activation M phase CDK activation
depends on the accumulation of M phase cyclin. In
the cell cycle of embryonic cells, cyclin is
consistently synthesized, and the concentration
of cyclin depends on the cyclin degeneration
speed. For the mitosis of most of cells, cyclin
is accumulated because of the enhanced
transcription of the genes of G2/M-cyclin and
M-cyclin. With the accumulation of cyclin,
the level of cyclin combined M-CDK(CDK1)is
increased. But the increased cyclin combined
M-CDK has no activity because Wee1 kinase has
phosphorylated the Thr14 and Tyr15 of CDK1 to
maintain the accumulation of CDK-cyclin. The
accumulated CDK-cyclin can be suddenly released
out to meet the needs of cell cycle. In M
phase, the decreasing of Wee1 activity and the
dephosphorylation of CDK by cdc25 are beneficiary
to CDK activation. Cdc25 can be activated by polo
kinase and M-CDK self (cdc25). Activated M-CDK
can inhibit the Wee1, the M-CDK inhibitor. So, a
feedback cycle will be formed. By this feedback
cycle, if a little bit CDK was activated by cdc25
or polo kinase, much CDK will be activated
immediately. The activation of CDK
needs its Thr161 phosphorylated by CDK activating
kinase (CAK).
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The activation of CDK1 needs its Thr14 and Tyr15
dephosphorylated and Tyr161 phosphorylated
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Check point of cell cycle Cell cycle is
exactly regulated by many check points. If the
one of the follows is happened, the cell cycle
will be stopped immediately DNA damaged, DNA
replicated incompletely, or spindle formed
incorrectly. Check point is composed of
the detector for abnormal events, signal pathway,
and effector. The main sites to be checked
include (1) G1/S check point It is
named start point in yeast and R point
(restriction point) in mammalians. This check
point controls the G1 cell turns to S phase. It
checks if DNA is damaged If the outer cell
environment is good If the cell size is
enlarged. (2) S check point It checks
if DNA is replicated completely. (3)
G2/M check point This check point controls cell
division. It checks if DNA is damaged and the
cell size is large enough. (4)
Meta-anaphase check point (Spindle assembly check
point) Any incorrect linkage of
centromere/kinetochore to spindle will inhibit
APC and stop the cell cycle.
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Four major check points
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Ataxia telangiectasia-mutated gene (ATM)
is an important gene associated with DNA damage
detection. 1 of humans are the heterozygote with
ATM absence. These persons are sensitive to
ionizing radiation and easy to suffering from
cancers. For the normal cells, the DNA damaged by
ionizing radiation will start the DNA repairing
mechanism, if the damaged DNA can not be repaired
the cell will be introduced to apoptosis. Anyway,
normal cells will not turn to mutated cells
(cancer cells) at this time. ATM encodes a
protein kinase that can bind to damaged DNA and
phosphorylate some proteins to stop cell cycle.
Two signal pathways are involved here (1)
Activate checkpoint kinase 1 (Chk1). Chk1
inhibits cdc25 and Ser216 by phosphorylating
them, and inhibits M-CDK by the phosphorylation
of cdc25. The cell cycle is stopped because M-CDK
was inhibited. (2) Activate checkpoint
kinase 2 (Chk2). Activated Chk2 can activate P53
by phosphorylating it. Activated P53 can cause
the expression of P21. P21 inhibits G1/S-CDK to
stop the cell cycle. The factors above,
such as P53 and P21, can be considered as tumor
suppressor genes. The factors above, like cdc25
or Ser216, can be regarded as tumor associated
genes.
56
Growth factor and cell proliferation In
our bodies, the following events about cell
proliferation must be controlled exactly. Which
type of cell should be proliferated? When they
will be proliferated? How much they should be
proliferated to? All of these events are depended
on the needs of body normally, and they are
controlled by the cell communication. In
unicellular organisms, all events above are just
depended on that the nutrition is enough or not.
Growth factors (GF) are the very important
signals associated with cell proliferation. So
far, over tens GFs have been found. Most of them
can promote cell proliferation, so, they are
called as mitogens, such as epidermal growth
factor (EGF), neuron growth factor (NGF), and
others. Some factors can inhibit cell
proliferation, such as chalone and tumor necrosis
factor (TNF). Transforming growth factor ß
(TGF-ß) is a very special one that takes
dual-directory regulation. TGF- ß can both
promote and inhibit different cell
proliferations. GF is formed and secreted
by adjacent cells. The molecule weight for each
GF is different, and most of GFs is composed of
one polypeptide chain, but some of GFs are
composed of two polypeptide chains, such as NGF,
TGF-ß, and hepatic growth factor (HGF).
The signal pathways for GF include ras pathway,
cAMP pathway, and phosphatidylinositol pathway.
If ras pathway is activated by GF, MAPK will be
activated. Activated MAPK will enter cell and
promote the expression of cell proliferation
associated genes. If c-myc is activated by some
unknown pathway, as a transcription factor, it
promote the expression of G1/S associated genes,
such as cyclin D, SCF, and E2F. Cell will turn to
G1 phase.
57
The function of GF
58
All events happened in cell cycle are carried out
like dominoes