L1 Cancer Genetics Regulated and unregulated cell proliferation

Question 1

Homeostasis: overview (5)

Answer 1

1. Cancer occurs in higher eukaryotes.
2. Cell survival and proliferation are highly regulated by integrated controls that continually evaluate the state of the cell and its environment.
3. NORMAL CELL PROLIFERATION IS MODULATED BY THE REGULATION OF THE CELL CYCLE.
4. Apoptosis eliminates damaged cells and cells needed only temporarily during development.
5. The accumulation of mutations in cancer cells allows them to escape apoptosis and proliferation controls.

Answer 2

a. Normal Cell: Death mechanisms offs, division checkpoints okay…
- Proper external cues for normal cell survival & proliferation
- no Death cues
- no growth- inhibition cues
- SURVIVAL CUES
- PROLIFERATION
= SURVIVAL AND PROLIFERATION

b. Normal Cell: Death programs activayed, Division checkpoints blocked.
- Proper external cues for normal cell survival & proliferation
- Death cues
- growth- inhibition cues
- no SURVIVAL CUES
- no PROLIFERATION
= CELL DEATH

c. Normal Cell: Death mechanisms offs, division checkpoints okay…
- Proper external cues for normal cell survival w/out proliferation (normal resting cell)
- no Death cues
- growth- inhibition cues
- SURVIVAL CUES
- no PROLIFERATION
= SURVIVAL BUT NO CELL DIVISION

d. CANCER CELL:
- no death cues
- survival cues
- proliferation cues
- no growth inhibition
= UNCONTROLLED SURVIVAL AND PROLIFERATION
- Self generated survival and proliferation signals in cancer cells

Question 3

Homeostasis: the role of Cell proliferation

Answer 3

DEVELOPMENT, HOMEOSTASIS, DIFFERENTIATE TO REPLACE DEAD CELLS, MITOSIS

1. Proliferation, ie cell progression through the cell cycle, is required for development AND homeostasis
2. Stem cells proliferate and differentiate in response to local signals to replace dead cells
3. (Asymmetric mitosis allows the stem cell population to replenish itself too)

Stem cell = stem cell and stem cell

Cancer:
Stem cell = stem cell and cell (Cell goes on to differentiate)

Question 4

Homeostasis: the role of apoptosis (cell death)

Answer 4

One of the roles of apoptosis is to SURVEY for CELLULAR ABNORMALITIES = EXECUTE SLEF DESTRUCT

Question 5

which cells experience apoptosis?

Answer 5

OLD ADULT CELLS, LOW TURNOVER, CELL DEATH.

1. Adult tissues = differentiated cells, plus some stem cells
2. Constant low-level turnover of cells: (Cells die and are replaced)
3. Cell death = accidental (necrosis) OR programmed (apoptosis)

• Eg. Cell is abnormal (dividing too rapidly or infected by virus), or no longer functional.

**Cell loss is not a problem as long as cell population is replenished (homeostasis)

Question 6

Homeostasis: how it is linked to cancer: 3

Answer 6

CLONE OF SOMATIC CELLS - HOMEOSTASIS ENCODING GENE MUTATED - ACCUMULATION OF MUTATIONS = CANCER

1. Most cancers develop through the accumulation of mutations in a clone of somatic cells (mutations that are not passed on)
2. The mutations generally occur in genes encoding molecules involved in homeostasis
3. Thousands of such genes and mutations leading to cancer formation have been identified

Question 7

Cell proliferation: Cell cycle
10.

Answer 7

1. During G1, the cell grows.
2. Cells may enter G0, a non-dividing phase.
3. After the G1/S CHECKPOINT: the cell is committed to dividing.
4. S PHASE - DNA duplicates
5. In G2, the cell prepares for mitosis.
6. After the G2/M CHECKPOINT, the cell can divide.

7.G0 - G2/M CHECKPOINT IS INTERPHASE

8. M PHASE: Nuclear and cell division.
   Mitosis
   - Mitosis and cytokinesis (cell division) take place in M phase.
9. Spindle Assembly checkpoint
10. Cytokinesis
    = 2 daughter cells
    cycle repeats.

Question 8

S, G2 and M fixed duration?

Answer 8

S, G2 and M phases are normally fixed duration

but G1 can be variable as cell cycle can enter optional G0 resting phase.

Question 9

Some cells have no G0 Phase?

Answer 9

Some cells, rapidly dividing (eg embryo) have no G0 phase.

Some differentiated
cells are in G0 for remainder of their natural lives

Question 10

Stem cells and Cell cycle?

Answer 10

Stem cells fluctuate between G0 and the cell-division cycle.

Question 11

What does the
G1/S CHECKPOINT
and
G2/S CHECKPOINT do?

Answer 11

G1/S Checkpoint
– Is the cell big enough?,
- Is environment favorable?
- Is DNA damaged?

G2/S Checkpoint
– Is DNA replication complete?

Question 12

CDK name and What is it? (2)

Answer 12

Cyclin-dependent protein kinases

– phosphorylates serine or threonine on target protein

– Generally present throughout the cell cycle

Question 13

Cyclin (4)

Answer 13

– A partner protein for CDK that dictates the substrate
specificity of the CDK

– unstable, therefore transient activity

– Present only in specific phase of cell cycle

– Each cyclin is expressed as a result of the activity of the
preceding CDK-cyclin complex (that activates a
transcription factor to express the new cyclin)

Question 14

What do CDK-cycline complex do?

Answer 14

Sequential activation of different CDK-cyclin complexes controls
cell cycle progression.

If an active CDK-cyclin is present at wrong time, it will cause inappropriate genes to be transcribed.

Question 15

Cell proliferation: Cell cycle regulation through CDK Cyclin complexes

Answer 15

1. Going to the next step in
   the cycle requires
   activation of genes whose
   protein products (cyclins)
   are necessary for the next
   phase of the cell cycle. (from G0 onwards..)

Variations in CDK-cyclin
activities throughout the
cell cycle in mammalian
cell.

Widths of bands indicate the relative activities of CDK-cyclin complexes.

CDK – Cyclin-dependent protein kinases

Cyclin – Concentrations vary in cyclical fashion

Question 16

Cell proliferation: Cyclin chooses the target
protein to be phosphorylated by CDK….EXPLAIN

Answer 16

1. Cyclin tethers target protein so that CDK can phosphorylate it.
2. CDK present throughout cell cycle, so which complex is active is a function of which cyclin is present.
3. As different cyclins present at different stages of cell cycle, each phase is characterised by phosphorylation of different target proteins.

1. Cyclin-CDK Binding
2. Active cyclin-CDK complex
3. Target protein binding
4. Phosphorylation of target protein.

Question 17

Cell proliferation: How do CDK Cyclin complexes control the cell cycle? (2)

Answer 17

1. CDK-cyclin complexes turn transcription factors ON AND OFF THROUGH PHOSPHORYLATION.
2. Either the Transcriptional Factor is phosphorylated, or a regulatory factor for the
   phosphorylated

Question 18

Cell proliferation: How does the phosphorylation
of some target proteins control the cell cycle?

Example 1: G1-S transition

Answer 18

1. Moving from G1 to S requires ‘gearing up’ for replication of the genome
2. The G1 CDK-cyclin complex phosphorylates many factors

-Transcription factors that turn on genes encoding:
* DNA polymerase
* Enzymes that produce deoxyribonucleotides
* Proteins involved in duplication of chromosomes
* Subunits of the next CDK-cyclin complex

-Regulatory factors that allow transcription factors to be active
* Rb-E2F pathway

Question 19

The G1 CDK-cyclin complex phosphorylates many factors - 2

Answer 19

-Transcription factors that turn on genes encoding:

• DNA polymerase
• Enzymes that produce deoxyribonucleotides
• Proteins involved in duplication of chromosomes
• Subunits of the next CDK-cyclin complex

-Regulatory factors that allow transcription factors to be active

• Rb-E2F pathway

Question 20

Cell proliferation: How does the phosphorylation
of some target proteins control the cell cycle?

Example 1: G1-S transition - Rb-E2F pathway

Answer 20

1. During G1, cyclin D and E levels rise and combine with associated CDKs,
2. by late G1 phosphorylation of inhibitor Rb (retinoblastoma protein)
   completed.
3. Phosphorylation
   inactivates Rb releasing E2F.
4. E2F is now free to transcribe genes that produce enzymes necessary for the replication of DNA.
5. AND transcribes the next cyclin, cyclin A, required for S phase.
6. Moves cells into S phase

Question 21

G1-S transition - Rb-E2F pathway

Answer 21

1. RB and E2F: RB binds to E2F and keeps it INACTIVE.
2. Cyclin-D-CDK and Cyclin-E-CDK —Phosphorylation.
   - INCREASING concentrations of cyclin-D-CDK and cyclin-E-CDK phosphorylate RB,…
3. Phosphorylation Inactivates RB, and it releases E2F.
4. E2F binds to DNA and stimulates the transcription of genes required for DNA replication.
5. Transcription to RNA

Question 22

Cell proliferation: How does the phosphorylation of
some target proteins control the cell cycle?

Example 2: G2-M transition

5 STEPS

Answer 22

1. Cyclin B combines with
   CDK to form inactive
   complex: Mitosis promoting
   factor (MPF)
2. MPF must be activated by
   dephosphorylation.
3. End of G2 MPF is activated which commits the cell to divide.
4. Active MPF phosphorylates
   other proteins which activate
   events associated with mitosis
5. At end of Metaphase, cyclin B is degraded, lowering amounts of
   MPF, return to interphase
   conditions

Question 23

‘MPF’ is a historical relic:

Answer 23

• it was identified before CDK and cyclins were identified, it is actually a CDK1-cyclinB complex

– see how its activity closely follows cyclin B levels..

Question 24

DNA damage inhibits the
activation of MPF!

Answer 24

DNA damage inhibits the
activation of MPF!

Question 25

Cell proliferation: How does the phosphorylation
of some target proteins control the cell cycle?

*G2-M transition in DETAIL 6

Answer 25

1. Cyclin B accumulates throughout interphase.
2. near the end of G2, active MPF reaches a critical level = causes the cell to progress through the G2/M checkpoint and into mitosis.
3. Degradation of cyclin B near the end of mitosis causes the active MPF level to drop = CELL REENTERS INTERPHASE.
4. Increasing levels of cyclin V during interphase combine with CDK to produce increasing levels of INACTIVE MPF.
5. Near the end of interphase, ACTIVATING FACTORS remove phosphate groups from MPF, producing ACTIVE MPF,
   - brings about the breakdown of the nuclear envelope, chromosome condensation, spindle assembly, and other events associated with Mitosis.
6. Near the end of Metaphase cyclin B degradation lowers the amount of ACTIVE MPF, which brings about ANAPHASE, TELOPHASE, CYTOKINESIS, and eventually Interphase.

Question 26

Cell proliferation: Checkpoints as brakes on the cell cycle EXPLAIN:

Answer 26

ORDER, COMPLETION OF EACH STEP, ACTIVATION OF PROTEINS

1. Proper progression through cell cycle is critical

eg Cannot go into M before DNA completely replicated,

2. Cell cycle checkpoints prevent progression until preceding stage has been successfully completed.
3. Activate proteins that inhibit kinase activity of CDK-cyclin complex ….
   Cell cycle is held in check until cell is properly
   prepared to proceed to next phase

Question 27

Cell proliferation: What is checked at ‘checkpoints’?

3 checkpoints PASS VS FAIL

Answer 27

1….G1/S CHECKPOINT:
PASS:
- Sufficient number of organelles
- growth factors activation

FAIL:
- Presence of TGFB proliferation inhibitor
- ATP deficiency

2…. G2/M CHECKPOINT:
PASS:
- Completely replicated genome
- large cell volume

FAIL:
- DNA damage

3….M/G1 CHECKPOINT
PASS:
-equal distribution of chromosomes between new daughter cells.

FAIL:
- Chromatids are not properly assembled on mitotic spindle.

Question 28

Cell proliferation: Checkpoints as brakes on the cell cycle.

p53

Answer 28

1… When DNA is damaged during G1, CDK-cyclin complexes stop
phosphorylating target proteins.

2. …P53 (“the guardian of the genome”) recognises DNA
   mismatches,

3… then proceeds to activate another protein, p21.

4…p21 binds to CDK-cyclin complex, inhibits protein kinase activity.

5. CDK cannot
   phosphorylate target
   and cell cycle unable to
   progress from G1 to S.
6. Once DNA repaired,
   p53 levels drop, p21
   levels drop, CDK-cyclin
   no longer inhibited, G1
   to S block is removed.

SLIDE 22

G0 PHASE:
- DNA DAMAGE
-p53
- P21
WHICH MEANS NOT RB-E2F;
RB Inactivation through phosphorylation and then E2F to DNA to enzymes for DNA synthesis to S phase,,,no cyclin-D-CDK nd Cyclin-E-CDK

Question 29

Apoptosis: ‘Programmed Cell Death’ EXPLAIN IN MULTICELLULAR ORGANISMS

• SELF DESTRUCT

Answer 29

In multicellular organisms, systems have
evolved to eliminate damaged cells
***- self
destruct mechanisms call PCD or apoptosis.

This self destruct mechanism can be activated under many different circumstances eg cells that are no longer needed for development.

Question 30

Apoptosis: 2 TYPES

Answer 30

1. Sequential destruction of cell
2. Driven by activity of caspases

Question 31

Apoptosis: Overview -Sequential destruction of cell (3)

Answer 31

1. fragmentation of chromosomes
2. organelle disruption
3. fragmentation of cell

Question 32

Apoptosis: Overview -

Driven by activity of caspases (4)

Answer 32

1. cysteine-containing aspartate-specific proteases
2. normally inactive as procaspases (precursor,
   harmless to cell)
3. Procaspases are activated by cleavage active caspases target other proteins for destruction
4. Caspases also cleave Dnase, leading to breakdown
   of cellular DNA and eventual cell death

Question 33

Apoptosis: Sequence of events

Answer 33

1. DNA is degraded
2. Cytoplasm nucleus skrink; nucleus fragments
3. BELBBING OCCURS:
   - the cell membrane bulges outward and breaks off into vesicles, taking some cytoplasm with it.

• Vesicles and the cell are engulfed by a macrophage.

4. The macrophage phagocytizes the apoptotic cell.

-phagocytosis by other cells
without any leakage of cellular contents or damage to nearby cells

Question 34

Apoptosis:
Two classes of caspase enzymes

Answer 34

Two classes of caspase

-Initiators and Executioners

Question 35

Apoptosis:
Two classes of caspase enzymes…

EXPLAIN INITIATOR VS EXECUTIONERS

Answer 35

1. INITIATOR caspase
   (eg caspase 8, 9) cleaved in
   response to activation signals
   (eg DNA damage, extracellular death signals).
2. They in turn cleave one of the EXECUTIONER caspases,
   which in turn cleaves another until all active …
3. PCD is mediated by a sequential cascade of
   proteolytic events that activate enzymes that destroy several key targeted cellular components.

Question 36

APOPTOSIS: HOW DO EXECUTIONER CASPASES WORK:

EG 3,6,7

Answer 36

1. Inactive executioner caspase
2. Cleaved by another caspase
3. Cleaved fragments = Large subunits, small subunit
4. Aggregation of executioner caspase subunits.
5. ACTIVE EXECUTIONER CASPASE
6. proteolysis of target proteins

which goes to either

7. Inactivation of DNA endonuclease-sequestering protein.
   = DNA fragmentation
8. ACTIVATION OF ACTIN-cleaving protein = Loss of normal cell shape.
9. OTHER TARGETS
   - Fragmentation of cell
   - Breakdown of organelles.

Question 37

Signaling: Controlling the balance of
Proliferation and apoptosis

Answer 37

• Cell: Are conditions appropriate to proceed in cell cycle, or commit suicide?
• Intracellular signals
  – cell cycle negative controls: inhibition of CDK-cyclin
  – cell cycle positive controls: activation of CDK-cyclin
  – apoptosis negative controls: maintain system in “off” mode
  – apoptosis positive controls: leakage of cytochrome c
  from defective mitochondria
• Intercellular signals
  – based on cell-cell communication
  – secreted molecules
  – direct cell-cell contact

Question 38

Signaling: Controlling the balance of
Proliferation and apoptosis

WHAT ARE THE INTRACELLULAR SIGNALS? = 4

Answer 38

1 – cell cycle negative controls: inhibition of CDK-cyclin

2 – cell cycle positive controls: activation of CDK-cyclin

3 – apoptosis negative controls: maintain system in “off” mode

4 – apoptosis positive controls: leakage of cytochrome c
from defective mitochondria

Question 39

Signaling: Controlling the balance of
Proliferation and apoptosis

WHAT ARE THE INTERCELLULAR SIGNALS? = 3

Answer 39

1 – based on cell-cell communication

2 – secreted molecules

3 – direct cell-cell contact

Question 40

Signaling: 2 Modes of intercellular signaling
EXPLAIN

Answer 40

A. Endocrine signals enter the circulatory system and can be received by distant target cells

• hormone secretion into blood by endocrine gland…blood vessel…distant target cell

B. Paracrine signals act locally and are received by
nearby target cells

• Secretory cell to Adjacent target cell

Question 41

Signaling: Two different types of signaling receptors

EXPLAIN THE PROCESS (5) ENDOCRINE

Answer 41

1. G-protein receptor that
   passes through the
   cell membrane ***seven
   times.
2. Enzyme-linked
   receptors.
3. Receptor tyrosine kinase, single transmembrane domain.
4. Active site of tyrosine kinase is in cytoplasmic domain.
5. Phosphorylation of
   tyrosine sidechains on
   target proteins.

Question 42

UNDERSTANDING Intercellular signals (3)

Answer 42

1. Protein ligands released in a tissue may act as
   signals that regulate cell division or apoptosis
2. There are pathways for controlling cell
   proliferation and self destruction.
3. Activation of pathways requires correct array of
   positive signals and absence of negative signals

Question 43

Signaling: Signals that promote proliferation

Answer 43

Cell cycle positive signals

Question 44

Cell cycle positive signals = 3

Answer 44

1 * Mitogens (growth factors) promote cell
proliferation and cell cycle progression

2 * Mitogens are protein ligands released from
paracrine sources

3 * Some mitogens activate receptor tyrosine kinase, initiates signal transduction pathway that leads to expression of G1 cyclin D genes

– Eg Epidermal growth factor, signals through
EGFR

Question 45

Signaling: Signals that prevent proliferation

Answer 45

Cell cycle negative controls

Question 46

Cell cycle negative controls = 2

Answer 46

Protein ligands inhibit cell division

– No need for growth

• Eg. TGF-beta, a ligand secreted under growth inhibitory conditions.

– binds its receptor..

– initiates signaling cascade which eventually blocks
phosphorylation of Rb

Question 47

RECALL: Rb regulates cell cycle by preventing activation of E2F TF
SO….

Answer 47

blocking Rb inactivation
keeps E2F off….DNA replication cannot proceed, cell cycle switched off

Question 48

Signaling: Signals that trigger apoptosis

Answer 48

Apoptosis positive controls

Question 49

Apoptosis positive controls explain = 2

Answer 49

1 * Often signal from neighbouring cell

– eg immune system cells; only small percentage cells
allowed to become functional, rest eliminated by
apoptosis.

– In developing fetus

2 * The self-destruct signal is activated through Fas
system and other death receptors.