Mod8 - Cycles of Division and Growth in Cell Populations Flashcards
Name 4 reasons why cells divide (mentioned)
Sustain life, propagate heritable traits, generate mass, generate diversity
Approximately how many cells - and cell types - are in the human body?
37 trillion (200 types)
What is the difference between embryonic and adult cell cycles?
Embryonic cell cycles can be simply S->M oscillators; adult stem cells have Gap phases to separate alternate S and M phases
What is G0 (name and function)?
QUIESCENCE: A dormant state which some cells remain in for years until they are ready to divide
Which Cdk is active in G1 phase, and which cyclin partner activates it?
Cdk4 AND Cdk6 (Cyclin D)
Which Cdk is active in G1->S phase, and which cyclin partner activates it?
Cdk2 (Cyclin E!)
Which Cdk is active in S phase, and which cyclin partner activates it?
Cdk2 (Cyclin A)
Which Cdk is active in M phase, and which cyclin partner activates it?
Cdk1 (Cyclin B)
What happens to Cdk and Cyclin levels throughout the cell cycle?
Cdk levels remain constant throughout, but Cyclin levels rise and fall to activate various Cdks as required
How do Cyclins actually activate Cdks?
Without a bound cyclin, the Cdk is inactive because the T-loop (or activation loop) occludes the active site;
When a cyclin binds to its partner Cdk, a conformational change pulls the T-loop away from the active site
Name 4 ways in which cyclin-Cdk complexes are regulated
- Phosphorylation (can be activating OR inhibitory)
- Ubiquitin-mediated proteolysis (provides directionality as you can’t un-degrade a cyclin)
- Localisation (e.g., cyclin B imported into nucleus JUST before mitotic entry
- CKI binding (Cdk inhibitors UPregulated in response to damage)
What could happen if the cell cycle progressed without the cell cycle checkpoints?
Progression without accurately duplicating and separating the chromosomes could lead to a damaged genome
Describe the 1st cell cycle checkpoint
Before entering S phase (end of G1) - “is environment favourable?” if not, Cdk4/6 activation is blocked
Describe the 2nd cell cycle checkpoint
Before entering mitosis (end of G2) - is all DNA replicated/is all DNA damage repaired? If not, Cdk1 activation is blocked
Describe the 3rd cell cycle checkpoint
Before duplicated chromosomes are pulled apart - are all chromosomes properly attached to mitotic spindle? If not, Cyclin B degradation is blocked
State the 4 steps required for “getting into and out of mitosis”
Step 1 - Switch off DNA damage checkpoints
Step 2 - Activate Cdk1 to get into mitosis
Step 3 - Silence the spindle assembly checkpoint (SAC)
Step 4 - Inactive Cdk1 to get out of mitosis
Describe how Cdk1 is activated (in step 2 of GIAOOM)
- First, Cyclin B must bind to Cdk1 (obvs)
- Then, it must be activated by Cdk-Activating Kinase (CAK), by phosphorylation
- THEN, it is INactivated by Wee1 kinase via phosphorylation elsewhere on the enzyme
- Finally, it is activated by Cdc25 which removes the inhibitory phosphates (Mitosis can now begin)
Why is there such an apparently convoluted mechanism for Cdk1 activation?
In reality, it is more of an equilibrium than linear, due to feedback loops; the active form of Cdk1 further activates Cdc25 (positive feedback) and inactivates Wee1 until eventually, all the Cdk1 is in the active form
How does Cdk1 actually trigger mitosis
It phosphorylates various target proteins, including other kinases, which activate mitotic pathways and inactivate interphase pathways; this brings about the morphological changes associated with mitosis (e.g., spindle assembly, chromosome condensation)
How do cells have “permission” to pass the G2/M checkpoint (in Step 1 of GIAOOM)?
Unreplicated or damaged chromosomes prevent entry into Mitosis, as repair pathways activate Wee1;
Once damage is all repaired, this pathway becomes silenced and equilibrium is balanced, so cell can enter mitosis
How is Cdk1 inactivated (in Step 4 of GIAOOM)?
Ubiquitylation and subsequent proteolysis of Cyclin B inactivates Cdk1
Why is a “less straightforward” method used in Step 4 to inactivate Cdk1, rather than just inhibitory phosphorylation?
Because DIRECTIONALITY! (Destruction of Cyclin B via proteolysis is irreversible)
Which protein is mainly responsible for Ubiquitylation and Proteolysis of Cyclin B to exit mitosis
APC acts as the E3 ubiquitin ligase (which actually joins ubiquitin to Cyclin B)
Why must the APC be carefully regulated (i.e., why can’t it be active all the time)?
If it were always active, it would constantly degrade Cyclin B, and mitosis would never occur
Describe the Silencing of the SAC (in Step 3 of GIAOOM) - and the role of the APC
The APC becomes active during mitosis, however the Spindle Assembly Checkpoint (SAC, which is activated by unattached kinetochores) blocks FULL activation of APC until chromosomes are all aligned;
Once kinetochores are attached, SAC is silenced, APC is activated
APC degrades Cyclin B to trigger mitotic exit (APC also degrades securin, triggering separation of sister chromatids -> so anaphase onset and mitotic exit are coupled)
What is erythropoietic (EPO) and what does it do?
It is a secreted cytokine that stimulates RBC production by activating the JAK/STAT pathway
Name the three (mentioned at start) diseases which can result if proliferation is not controlled
Colon cancer; leukemia; gingival hyperplasia
Are proliferation and growth the same thing?
NO - proliferation is the division of cells to increase in number; cells can grow without proliferating (e.g., large neurons) due to increased protein synthesis and decreased degradation
Name the four main types of external factors that affect proliferation/growth etc in cells
Mitogens - stimulate proliferation (by driving cell cycle)
Growth factors - stimulate growth
Survival factors - suppress apoptosis
Differentiation factors - control cell lineage
Name the four main types of external factors that affect proliferation/growth etc in cells
Mitogens - stimulate proliferation (by driving cell cycle)
Growth factors - stimulate growth
Survival factors - suppress apoptosis
Differentiation factors - control cell lineage
What is the restriction (R) point?
The “point of no return” in the cell cycle - up until this point, cells are responsive to mitogenic GFs and to TGF-ß, but past the R point, the cell is committed to completing the cycle
What is the R point in terms of Cyclins?
R marks the handover from Cdk4/6 to Cdk2 (driven by the rise of Cyclin E)
How does Cyclin D rise in early G1?
Mitogens activate RTKs -> Ras GTPase -> MAPK pathway (Mitogen activated protein kinase) -> transcription factors -> Cyclin D
How does the rise of Cyclin D lead to the rise of Cyclin E in late G1?
Cyclin D-Cdk4/6 complex phosphorylates Rb - a transcriptional repressor normally bound to E2F - causing it to release E2F
E2F then drives synthesis of Cyclin E, which then activates Cdk2, driving the G1/S transition
Name 3 different pathways that feed into Cyclin D (note that there are other examples too)
Growth factors in breast -> Herceptin -> pathway
Wnts in intestine
EPO cytokine pathway
Describe how E2F drives its own synthesis
Positive feedback: Cdk2 phosphorylates Rb, driving further rise in Cyclin E, until the G1/S transition
What is one (mentioned early) example of how CyclinE-Cdk2 activity drives entry into S phase
Phosphorylating replication origins, thus triggering DNA synthesis
Describe the role of proliferation in the small intestine
Paneth cells at the base of the crypt secrete Wnt signals, which are received by stem cells, causing them to induce cyclin D and proliferate
Why do not all cells in the small intestine proliferate?
Precursor cells migrate up and out of the crypt (away from the Wnt) signals
Once out of range, they stop proliferating and then differentiate
What are the two main types of carcinomas?
Adenomas (benign) and adenocarcinomas (malignant)
State the stages from normal epithelium to metastasis (cancer)
Normal Epithelium -> Hyperplastic Epithelium -> (early/intermediate/late) adenomas -> carcinoma -> invasion and metastasis
Describe what is meant by ‘Clonal Expansion’
One cell in a layer mutates, giving it a proliferating advantage
Then, a specific clone within this layer becomes dominant due to a second mutation
Eventually, there may be mutations which increase the mutation rate, leading to multiple parallel clonal expansions
Name the 4 hallmarks of cancer focused on in this lecture
- Sustaining Proliferative Signalling
- Evading Growth Suppressors
- Resisting Cell Death
- Genome Instability + Mutation
What is the fundamental cause of cancer?
Mutations in oncogenes and tumour suppressor genes (which together control proliferation, differentiation and survival of cells), leading to abnormal cell behaviour
How do mutations in oncogenes and tumour suppressor genes “manifest themselves” in tissues?
They manifest as deregulated tissue homeostasis, in turn disrupting normal tissue architecture
What was the first oncogene to be discovered, and how did it come about?
src -> first observed in Rous Sarcoma Virus (RSV)
Most retroviruses only had 3 genes, but RSV also had v-src, which had a cellular counterpart c-src.
c-src regulates a signalling pathway for the cell cycle; at some point, a pro-virus got accidentally integrated next to c-src and then got taken up with it, causing src to become part of the viral genome
Define viral oncogenes
Mutated versions of cellular genes that regulate proliferation, found in viral genomes
State the common Ras mutation commonly associated with cancer, and how this works
Ras G12->V (this blocks Ras’ ability to inactivate itself via GTP hydrolysis to GDP. Therefore, Ras stays active and drives Cyclin D synthesis even in the absence of mitogens (NOTE - ONLY A SINGLE MUTATED RAS ALLELE REQUIRED FOR THIS)
Why does mutation of (both) Rb alleles lead to Retinoblastoma?
In the absence of Rb function, E2F is constitutively active, and will drive entry into S-phase even in the absence of mitogens
Why is familial retinoblastoma far more common than sporadic?
Both Rb copies must be mutated for disease; if one copy is already mutated (inherited), then it is quite possible for the other to mutate as well. However, it is very unlikely for both copies to mutate by chance
How does Neurofibromatosis arise?
If both copies of the NF1 tumour suppressor gene are mutated and NF1 function lost, Ras is constitutively active, causing cancer
What happens inside cells in the small intestine when Wnt signals are not present?
A 3-protein complex forms [active GSK-3ß + APC + ß-catenin]. Active GSK-3ß phosphorylates ß-catenin, causing it to be degraded and no Cyclin D to be produced
What happens inside cells in the small intestine when Wnt signals ARE present?
GSK-3ß is inactivated, so ß-catenin is NOT phosphorylated and degraded; ß-catenin translocates to the nucleus, where it stimulates Cyclin D synthesis, causing proliferation
What are APC and ß-catenin examples of, respectively?
Tumour suppressor gene + Oncogene
Why do ß-catenin mutations lead to colon cancer?
(NOTE: ONLY 1 DOMINANT MUTATION REQUIRED)
This can prevent phosphorylation + degradation of ß-catenin, so Cyclin D is continuously produced even in the absence of Wnt signals, leading to continuous proliferation of non-differentiating cells
(leading to Hyperplasia -> Polyp -> Adenoma -> Adenocarcinoma)
What is TFG-ß and what can happen if it is mutated?
It is an important tumour suppressor (“a brake on the cell cycle”) - mutation can permanently “release the brake” on the cell cycle
Name 3 examples of apoptosis in normal cell physiology
Tadpole tails during embryonic development; separating digits in mouse paw; eliminating immune cells which recognise “self”
Describe p53 concentration under normal conditions and how this is maintained
Normally low, because Mdm2 ubiquitinates p53, so it is proteolysed
How does p53 normally pause the cell cycle when the cell is under stress?
Stress inhibits Mdm2, so p53 is stabilised, p53 then activates p21, which inhibits Cdk2, thus preventing the transition to G2 phase
Distinguish between the intrinsic and extrinsic apoptosis pathways
Intrinsic: internal signals
Extrinsic: external signals
How do both the intrinsic and extrinsic apoptosis pathways actually cause cell death?
Activation of CASPASES, a family of proteases which lead to protein and DNA digestion
How are the mitochondria involved in apoptosis
Normally, cytochrome c is localised within the mitochondria. However, the formation of pores in the Outer Mitochondrial Membrane (due to pro-apoptotic signals) can allow cytochrome c to be released into the cytosol, leading to apoptosis
How does the release of cytochrome c into the cytosol lead to apoptosis?
Cyt c is a part of the APOPTOSOME that is assembled, with active caspase 9 at the centre
Active caspase 9 then causes proteolysis of death substrates (e.g., ICAD, lamin, vimentin and actin)
How does proteolysis of ICAD (one of the death substrates) lead to apoptosis?
ICAD is an inhibitor of CAD (Caspase-Activated DNase); when caspase degrades ICAD, CAD is free to digest DNA
How is p53 involved in the “fight to control the pore” between survival and death factors?
p53 inhibits pro-survival factors, and brings the balance more in favour of pro-death factors (p53 mutations can prevent apoptosis as pro-survival factors are not inhibited)
What is the role of BcI-xL and BcI-xLi in apoptosis?
BcI-xL is a pro-survival factor; the drug BcI-xLi inhibits it, thus enhancing Taxol activity and sensitising cancer cells to chemotherapy
Briefly describe the extrinsic apoptosis pathway
Death ligands bind to extracellular death receptors, which then activate initiator pro-caspases to active Caspase 8 and Caspase 10, which then activate executioner procaspases, which leads to proteolysis of death substrates
Name 3 death signals (extrinsic)
FasL, Tnf-alpha, Trail
What is the function of the Fas extrinsic apoptosis pathway?
Eliminating T cells that recognise “self”
If a T-cell binds an antigen-presenting cell recognising “self” then FasL is expressed, which then binds to its own Fas receptor, inducing apoptosis
What can happen if there are defects in the Fas pathway?
Autoimmune syndrome
Describe the impact of the MYC oncogene on cancer incidence in mice
Overexpressing the MYC oncogene alone did not cause cancer, as apoptosis balanced out the hyperproliferation
Only when BcL-xL is also activated does cancer occur, as hyperproliferation is no longer balanced by apoptosis
