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Importance of the cell cycle
• The identification of the factors that regulate the cell
cycle were discovered through the seminal work of 3
important scientists (Lee Hartwell, Paul Nurse and Tim
Hunt) who were awarded the Nobel Prize in Physiology
or Medicine in 2001.
Lee Hartwell,
S. cerevisiae
Paul Nurse,
S. pombe Tim Hunt,
Sea urchin
The Cell Cycle
• The cell cycle is a series of temporally
ordered events that leads a cell to divide
itself into two daughter cells.
• It is a complex system
• The components are identified
• It is a highly regulated process
• Although both somatic cells and germ cells
divide, they follow slightly different processes
named mitosis and meiosis. respectively. This
lecture focuses on the division of somatic cells, or
mitosis.
Cell Cycle
Mitosis
• Mitosis produces 2 daughter cells
• The two daughter cells containt the exact same
number of chromosomes as the original parent cell
• Daughter cells are DIPLOID (2N DNA content)
Wikipedia
Cell Cycle Characteristics
• Temporally ordered events
• It cannot go backwards
• Positive and negative feedback loops regulate
the function of the molecules involved and
ensure the coupling of the following phase on
the accomplishment of the previous
• Checkpoints maintain the order of the events in
case something goes wrong
When do cell numbers change ?
• Growth and development (expand pool of cells)
• Tissue turnover (replace lost cells e.g. blood loss)
• Response to injury (add new cells to wound, replace lost cells)
• Physiological changes (e.g. lactation, infection, hypoxia, exercise)
• Need to balance production with loss for tissue homeostasis
• Need to remove unwanted excess after demand is met
The cell cycle
• Is divided in 2 morphological and 4 biochemical stages
• Interphase – First gap G1
– Replication of chromosomes S
– Second gap G2
• Mitosis & Cytokinesis
– Mitosis M
Mitotic figures in tissues
8 months
2 mm
Pituitary tumors in 8-month-old mutant rats
2 mm
Scheme of the cell cycle
G0 is the 5th phase of the cell cycle
The Cell: A Molecular Approach, Sinauer Associates 2000.
G1 Phase
• Cells are metabolically active
• Organelle duplication, but no DNA replication
• Duration variable – short in embryonic and cancer cells
• Cell prepares for S phase
• Cells that remain in G1 for a long time = G0 (permanent tissues, such as neural tissue)
S Phase
• Committed to cell division once the S phase
starts
• DNA and centrosome replication
• Semi-conservative replication of DNA: two identical daughter genomes
Restriction of DNA replication
• DNA replication is restricted to once
per cell cycle
• MCM (mini-chromosome maintenance)
proteins bind to origins of replication
together with ORC (origin replication
complex) proteins, which initiate DNA
replication.
• MCM proteins only bind to DNA in G1
The Cell: A Molecular Approach, Sinauer Associates 2000.
G2 Phase
• Growth continues
• Enzymes and proteins for cell division are
synthesized
• Determining Cell Stage
– Cells at different stages of the cell cycle can also be
distinguished by their DNA content (in G2=4N)
Mitotic (M) Phase • Mitosis plus Cytokinesis:
– Mitosis - The division of the nucleus that results in
identical complete copies of chromosmes packaged
into two new nuclei
Cytokinesis - The division of the cytoplasm that
results in two daughter cells
• Mitosis is divided in 4 phases:
– Prophase
– Metaphase
– Anaphase
– Telophase
Mitotic (M) Phase-2
Analysis of cells replication status
G0/G1 : 34%
G2/M : 15%
S : 51%
• Cell cycle analysis by fluorescence-activated cell sorting (FACS)
http://www.icmb.utexas.edu/core/Microscopy/pdf/CELL_CYCLE_FUNDAMENTALS.pdf
The duration of the Cell Cycle
• Cell Cycle Lengths
– Vary by cell type:
• Embryonic cells
• Stem cells (e.g., blood cells and epithelial cells)
• Sperm cells
• G1 prolonged in stable or permanent cells (called G0)
• G1 rapid or non-existent in rapidly-dividing cells
Embryonic cells
• Have to divide rapidly
• All energy goes into DNA synthesis
• So G1 lacking and G2 quite short
• Each round of division subdivides original
cytoplasm into smaller and smaller cells,
• Until adult cell size is reached
Embryonic cells division
The Cell: A Molecular Approach, Sinauer Associates 2000.
Cell Cycle control: 3 levels
External control Hormones, Growth factors, Cytokines etc
G0
G1 G2
M
S
Intracellular control Signalling cascades coordinate nuclear and cytoplasmic response (MAP Kinases etc)
Intrinsic control Checkpoints within cycle to coordinate replication, repair, chromosome segregation & cytokinesis
What regulates cell cycle progression?
• Early experiments of cell fusions (1970, Rao & Johnson)
S-phase cell
G1 nucleus
S-phase induced in G1 nucleus
S-phase cell
G2 nucleus
G2 nucleus remains G2
• When G1 cells were fused with S phase cells, the G1 nucleus
immediately began to replicate DNA.
• When G2 cells were fused with S phase cells, only the S phase
nucleus continued DNA replication.
The cell cycle is coordinated by
diffusible factors
Facit: Cell cycle cannot go backwards!!!
• Based on the cell fusions it appears that the G2
nucleus has to pass through M and enter G1 before
another round of DNA replication could be initiated:
-coupling of S phase to M phase
-S-phase “factors” only work on G1 nucleus
What are these „diffusible“ factors?
• These factors are protein complexes
• The first components to be identified were
proteins that periodically appear in specific
phases of the cell cycle = cyclins
Cyclins
• Regulatory subunits of complexes with
kinase activity (with Cdks)
• Their presence “oscillates” in the cell cycle
• There are G1, S, G2 and M cyclins
Spindle-assembly checkpoint
Cycle transit marked by cyclical
appearance of Cyclin proteins
Cyclin D appears
Cyclin E appears
G1 phase S-phase
Temporal expression of cyclins
www.streaming.cineca.it
MPF : Mitosis-promoting factor= Cyclin-Cdk complex
MPF is a complex containing Cyclin B
and Cdk1
The cell cycle engines
• Cyclin Dependent Kinases (CDKs)
- Become active when bound to the regulatory subunit, the cyclin
CD
K
cyclin
substrate
ATP
P
product + ADP
Cyclin D-CDK4
Cyclin E-CDK2
Cyclin A-CDK2
Cyclin B-CDC2
Cyclin-CDK complexes
asyn 0 4 8 12 14 16 20 24 28 32 hours
cyclin A
cyclin E
Cyclin and CDK expression as cells re-enter the cell cycle
G0 G1 S cell cycle phases
CDK activity
Kinase activity of the CDK component
• The kinase activity of each cyclin/Cdk complex starts once the complex is assembled (=once cyclin is synthesized)
The cell cycle brakes
• Cyclin Dependent Kinases Inhibitors (CKIs)
Cyclin D-CDK4
Cyclin E-CDK2
Cyclin A-CDK2
Cyclin B-CDC2
CDK inhibitors
p27Kip1 p21Cip1
p27Kip1
p16INK4
p21Cip1
p18
p19
Cyclin Dependent Kinases Inhibitors (CKIs)
Mod. from van den Heuvel, Cell Cycle Regulation, Wormbooks.
Complexity of cell cycle regulation
Worm Mammals Cyclin D, Cyclin E 20 Cyclin A, B, C, D1, D2, D3, E, F, G, H (plus splice variants) Cdkk 1 Cdkk (Cdk7/CAK) Cdk2, Cdk4 7 Cyclin dependent kinases Cdk1-7 Cdki1, Cdki2 7 Cdk-inhibitors:
- CIP/KIP family p21, p27, p57 - INK4 family p15, p16, p18, p19
Regulated destruction of cell cycle
proteins
• A number of proteins are regulated by turnover:
proteolysis. Ensures that cell cycle can't roll
backward. This requires that the targets be
ubiquitinated by specific ubiquitn ligases, which
targets them to the proteosome for destruction.
– Degradation of cyclin is essential to keep cell cycle
moving forward. Making a cyclin mutant that cannot
be degraded traps cells in M phase.
Proteasome-mediated degradation • Removal of proteins when no longer needed or deleterious is
accomplished by the proteasome
Proteasome
www.ncbi.nlm.nih.gov/books/NBK9957
Regulation of G1/S progression
Cdk2
Cdk4,Cdk6
ubq
G0
G1 G2
M
S
E2F (active)
ubq
Cyclin D
Cyclin E
p21, p27
Rb E2F
p16
Cyclin D
Cdk4,Cdk6
Cdk2
Cyclin E
Rb
P P
Rb protein:
• Key substrate of Cdk-cyclin D complex
• Binds to transcription factor E2F
• Growth factors -> activated Cdk-Cyclin
• Phosphorylates Rb
• Allows transcription to proceed
• Rb gene: expresses Rb protein, acts as a G1
brake
Function of the Rb protein
Rb: a paradigm for tumor suppressor gene function
• Two classes of genes in cancer:
– Oncogenes
– Tumor suppressor genes
• Oncogenes-> positively regulate cell
proliferation (dominant mutations)
• Tumor suppressor genes -> normally
negatively regulate cell proliferation
(recessive mutations)
Rb: a paradigm for tumor suppressor gene function-2
www.ncbi.nlm.nih.gov/books/NBK13944
Inhibition of Rb function
• Checkpoints maintain the order of the cell
cycle events by signaling if something
goes wrong:
• e.g. DNA damage in G1 or G2, lack of nutrients
• incomplete DNA replication
• incomplete establishment of mitotic apparatus.
Cell-Cycle Checkpoints
Cell-Cycle Checkpoints
• G1 checkpoint
• In yeast, called start
• In animal cells, called restriction point
• G2 checkpoint
• Located at boundary between G2 and M phase
• Proper completion of DNA synthesis required before cell can initiate mitosis
• Spindle Assembly Checkpoint
• Boundary between metaphase and anaphase
• All chromosomes must be properly attached to the spindle
Cell cycle checkpoints-2
G1 checkpoint
• G1 Checkpoint:
– START in yeast
– Restriction Point in mammals
• Main step that commits a cell to division
• Sensitive to:
– Cell size
– Availability of nutrients
– External growth factors
• Cyclin D
– Sensitive to growth factors (rapidly degraded)
– Cdk-cyclin D complex drives cell through G1 to S phase
Restriction Point
The Cell: A Molecular Approach, Sinauer Associates 2000.
• The availability of growth factors controls cell cycle
progression in animal cells at a point in late G1 called the
restriction point.
• If they are not available, the cells enter a quiescent stage of
the cycle called G0.
Cellular response to DNA damage
A major player in the response to radiation-
induced DNA damage
Khalil et al. BioDiscovery 2012; 1:3.
G1 checkpoint
Rb
Rb-P
E2F
Damage / Stress ATR/ ATM
p53 Cdc25-P
Cdc25
Chk1 Chk2
Cdk2 Cyclin E
p21 IP ubq
Rb-P
E2F
Rb-P
Rb
Rb-P
E2F
Rb
E2F (active)
Rb-P
Elected „Molecule
of the year“ in 1993
• p53 is mutated in over 50% of all human cancers; when is
mutated it looses ist tumor suppressive function
p53: the guardian of the genome
• DNA damage induced by irradiation,
carcinogens etc. leads to rapid
increase in the amount of p53 protein.
• p53 is a transcription factor
• When activated, p53 induces the
transcription of the CKI p21 cell
cycle arrest at the G1 checkpoint
p53: the guardian of the genome
The Cell: A Molecular Approach, Sinauer Associates 2000.
• Following DNA damage, oxidative stress, ribonucleotide
depletion… p53 dissociates from its regulator Mdm2 and
becomes more stable
• Then it is phosphorylated by various kinases (depending
on the original stimulus) and this change in conformation
activates its transcriptional activity
Mechanisms of p53 activation
Gopalkrishnan et al.
J Biol Chem. 1998;273:10972-8
p53 is a transcription factor
Bykov VJ, et al. Eur J Cancer. 2003;39:1828-34.
• Upon stabilization, p53
activates genes involved
in cell cycle arrest or
apoptosis (pro-apoptotic)
p53: Tumor Suppressor Gene
• p53 induces a temporary cell cycle arrest in G1 to allow
the cells to repair their DNA
• If DNA damage is too extensive and cannot be repaired,
p53 induces apoptosis (programmed cell death)
• Mutations in p53 G1 checkpoint no longer works
cells can replicate damaged DNA and accumulate
mutations
Adre
nal cort
ical tu
mor
Mutant p53 (R337H) wt p53 Ribeiro et al. Proc. Natl. Acad.
Sci. USA 2001; 98: 9330–9335.
p53 Tumor Suppressor Protein
Triggers Cell Suicide
Normal cell Cell suicide
(Apoptosis)
p53 protein
Excessive DNA damage
Mitotic Cdk-Cyclin
Complex (MPF) and G2
• MPF= cyclin B/CDK1
• Controls G2 checkpoint by phosphorylating proteins involved in early stages of mitosis
• Cdk levels constant
• But mitotic cyclin levels gradually increase – act as cell regulators
• MPF only active when cyclin B levels high enough – triggers passage through G2 checkpoint
Function of G2 checkpoint
• Error check: DNA replication must be completed
• Detects unreplicated DNA, holds cell at
G2
• Detects damaged DNA, arrests cell in G2 until damage repaired
G2 / M checkpoint
Cdk 1
Cyclin B
Plk1, Aurora
Cdc25 Cdc25
P
Release to M (Prophase)
Cdc25-IP ubq
Cdk 1
Cyclin B
• In normal conditions the phosphatase Cdc25 is required for the cell to move into M phase
Moving into anaphase
Cdk 1
Cyclin B
Cdc25
P
Release to anaphase
ubq
Cdk 1
Cyclin B
•MPF (cyclinB/Cdk1) causes activation of anaphase-promoting
complex (APC), a regulatory pathway that promotes anaphase
• APC promotes the degradation of Cyclin B thereby inactivating
MPF and pushing the cell forward into anaphase
APC/C Cyclin B
Degradation
Regulation at spindle-assembly
checkpoint
• The spindle checkpoint prevents anaphase
onset until all chromosomes are properly
attached to the spindle.
• To achieve proper segregation, the two
kinetochores on the sister chromatids must be
attached to opposite spindle poles
Spindle-assembly checkpoint • The spindle checkpoint prevents anaphase onset until all
chromosomes are properly attached to the spindle.
• To achieve proper segregation, the two kinetochores on the
sister chromatids must be attached to opposite spindle poles
Spindle-assembly checkpoint • The target of the spindle checkpoint is Cdc20 which binds to APC/C
and confers specificty to its ubiquitin-depepdent degradation
• This complex targets securin for destruction, freeing saparase and
enabling the destruction of cohesins and thus sister chromatid
separation
Wikipedia
Cdc20
Degradation
Why understanding cell cycle
regulation is so important?
• Many of the genes mutated in human cancer are
directly involved in regulation of the cell division
cycle, because such genes are most intimately
linked to the machinery that controls cell
proliferation.
• A basic understanding of the machinery that
drives the cell division cycle (or cell cycle, for
short) is therefore indispensable for the study of
molecular oncology.
Cell cycle in normal cells
M (mitosis)
2 homologous pairs are shown
DNA repair
genes
Oncogenes
Tumor suppressor
genes
G2
G1 (cell growth)
S (synthesis)
Cell cycle genes can act as oncogenes
From proto-oncogene to oncogene
Mutated cell cycle genes
Normal genes
Oncogenes promote cell division (i.e. cyclins and CDKs)
T.s.g.
Tumor suppressor genes block cell division (i.e. CDK inhibitors)
T.s.g.
Mutation/loss of CDK inhibitors=accelerated cell division
Normal t.s.g. genes= Regulated cell cycle
Cell cycle genes can act as tumor
suppressor genes
Cell cycle control and cancer
• Cyclin D1 Amplification in ca 20% Mammary cancer
Translocation / amplification in 50% of lymphoma
• Cdk4 Overexpression in mammary cancer and glioblastoma
• Cyclin E Overexpression in 10% mammary cancer
• Cdc25 Overexpression in 20+ cancers
• p16 Loss (non-mutational) in many cancers
Mutation in inherited melanoma
• p27 Loss in many cancers
Mutation in inherited neuroendocrine cancers
• Rb1 Loss / mutation in cancers (retinoblastoma, osteosarcoma)
• Chk2 Mutation in Osteosarcoma
• When the G1 checkpoint misfuctions, this can
lead to duplication of damaged DNA:
accumulation of mutations
escape from apoptosis
Cell cycle control and cancer-2
Tumorigenesis
Khalil et al. BioDiscovery 2012; 1:3.
Cell cycle control and cancer-3 • When the spindle checkpoint misfuctions, this can lead to
chromosome missegregation and aneuploidy.
Tumorigenesis
Holland & Cleveland. EMBO reports, 2012; 13, 501-514.
Inactivation of key cell cycle regulators by
viral proteins • DNA tumor viruses transform cells, at least in part, by
inactivating tumor-suppressor gene products. • This further supports the important role of Rb and p53
proteins in cell cycle regulation.
www.streaming.cineca.it
Summary-1
• Most important characteristics of the cell cycle:
• Highly regulated
• Moves only forward
• Cyclin binding activates Cdk, which target transcription factors
to ensure cell cycle progression
• Checkpoints make sure that everything progresses in the
proper order and without mistakes
Summary-2
• Later steps can only occur if early steps are completed
• Cell cycle “engines” (cyclin/Cdk complexes) and inhibitors (CKIs)
• Periodicity of the availability of key molecules, through synthesis (cyclin) and ubiquitination + degradation (inhibitory proteins)
• Most, if not all, components have mutations in some cancers:
2 consequences: uncontrolled proliferation and lack of checkpoint control.
These lead to increased mutability and cancer progression.
