CANCER; Lecture 1, 2 and 3 - Cell pathology of cancer, Cell cycle and regulation, Signalling mechanisms in growth and division Flashcards
What is metaplasia?
Reversible change in which one adult cell is replaced by another adult cell type -> adaptive and it isn’t neoplastic
What is dysplasia?
An abnormal pattern of growth in which some of the cellular and architectural features of malignancy are present -> pre-invasive stage with intact basement membrane, so this is the stage at which cancers need to be caught as it doesn’t spread into lymphatics -> low-grade is reversible but high grade not really
Where are the common sites of dysplasia?
Cervix (HPV iinfection), bronchus (smoking), colon (UC), larynx (smoking), stomach (pernicious anaemia), oesophagus (acid reflux)
What are the cell characteristics of dysplasia?
Loss of architectural orientation, loss in uniformity of individual cells; nuclei are hyperchromatic/enlarged, mitotic figures are abundant, abnormal and in places where they aren’t usually found
What is a neoplasia, tumour or malignancy?
An abnormal, autonomous proliferation of cells unresponsive to normal growth control mechanisms
What is the difference between benign and malignant?
1) Don’t invade/met. 2) Encapsulated (leiomyomas are benign but not encapsulated). 3) Usually well differentiated. 4) Slowly growing. 5) Normal mitoses
When are benign tumours fatal?
What are the characteristics of malignant tumours?
What is metastasis?
A discontinuous growing colony of tumour cells, at some distance from the primary cancer
How does metastasis occur?
What is the nuclear-cytoplasmic ratio in well vs poorly differentiated tumours?
High in poorly diff. and low in highly diff.
What are the different kinds of tumours?
Benign epithelial tumours, carcinoma, benign soft tissue tumours, sarcoma, leukaemia & lymphoma, teratoma, hamartoma (localised malformation, not neoplastic)
What are benign epithelial tumours?
Of surface epithelium = papilloma (skin, bladder); glandular epithelium = adenoma (stomach, kidney, pit, thyroid, colon)
What are carcinomas?
A malignant tumour derived from epithelium
What are sarcomas?
A malignant tumour derived from connective tissue (mesenchymal) cells
What are leukaemias and lymphomas?
- Tumours of WBC;
- leukaemia = malignant tumour of bone marrow derived cells which circulate in the blood;
- lymphoma = malignant tumour of lymphocytes in lymph nodes
What are teratomas?
- Tumour derived from GERM cells, which has potential to develop into tumour of all 3 germ cells layers;
- Testes (mainly malignant) and ovaries (mainly benign) mainly;
- dermoid cysts = mature/benign teratomas in ovaries (have teeth, eyeball, brain, hair)
What are hamartomas?
- Localised overgrowth of cells and tissue native to the organ - No malignant potential;
- common in children and should stop growing when they stop.
How do you assess differentiation of tumours?
Evidence of normal function still present production of:keratin (squamous cells), mucin (Glandular epithelium), bile (hepatocytes), hormones
How do we differentiate tumours?
Grading - how much a tumour looks like tissue; staging (!!) - how far it has spread; also if aren’t differentiated then called anaplastic carcinoma
What is TNM?
NB: stage is so much more than grade
What is the Gleason grading system?
(Prostate)
What are the different cell division times in specific cells?
What is the relevance of appropriate regulation of cell division?
Contact inhib of growth = cells usually grow by sensing neighbouring cells -> tumours lack this
What is the cell cycle?
Orderly sequence of events in which a cell duplicates its contents and divides in two.
What is the regulated progression through the cell cycle?
Cells need to duplicate their genetic material with mitosis being the most vulnerable point in the cell cycle
Which is the most vulnerable period of the cell cycle and why?
Mitosis -> Cells are more easily killed (irradiation, heat shock, chemicals), DNA damage can not be repaired, Gene transcription silenced, Metabolism?
What is the eukaryotic cell cycle?
Cells normally rest in G0 and mitosis happens fast (5min)
What is the function of the S phase?
DNA replication; protein synthesis (initiation of translation and elongation increased (capacity increased); replication of organelles (centrosomes, mitochondria, golgi) in case of mitochondria, needs to coordinate replication of mDNA
What is the centrosome?
Organelle near the nucleus of a cell which contains the centrioles and from which the spindle fibres develop in cell division -> regulate microtubule network to orchestrate cell division
What is the life cycle of centrosomes during mitosis?
- Cell initiates duplication and enters the cell cycle, centrosomes duplicate ->
- mother and daughter will separate into a mother and daughter taking place in the S phase.
- Cloud of protein complexes surround them and make nucleating sites for the microtubules
What are the 6 different phases of mitosis?
What is prophase?
- Condensation of chromatin -> condensed chromosomes -> each consists of 2 sister chromatids, each with a kinetochore.
- Condensation of DNA occurs so you can minimise DNA damage during mitosis
- -> double helices wrapped around histones to form chromatin and further wrapped until forms chromosome
What is a kinetochore?
Complex of proteins which is a key regulator of processes around chromosomes in the cell cycle
What occurs in prophase?
- Replicated chromosomes condense,
- nuclear envelope breaks down so chromosomes move into cytoplasm,
- duplicated centrosomes (late prophase) migrate to opposite sides of the nucleus and
- organise the assembly of the microtubules;
- mitotic spindle form outside nucleus between 2 centrosomes -> centromeres act as a belt (constricts around chromosomes)
How does spindle formation occur?
Radial arrays meet in the middle (called polar microtubules), form highways letting chromosomes know where to go
What is metaphase?
Chromosomes leak into cytoplasm, following breakdown of nuclear envelope and go with their pairs to the centre of the cells
What occurs in early prometaphase?
One microtubule array will attach to the kinetochore on one side and another microtubule array will attach to the other side
What occurs in late prometaphase?
In kinetochore there is a specialised protein which sense attachment of microtubules (CENP-E)
What is anaphase?
Paired chromatids separate to form 2 daughter chromatids -> cohesin (protein complex) which holds sister chromatids tightly bound together
What occurs in anaphase A?
- Cohesin broken down and microtubules get shorter,
- with chromatids moving towards centrosomes;
- daughter chromatids pulled towards opposite spindle poles
What occurs in anaphase B?
Daughter chromosomes reach opposite poles either by shortening of microtubules that form the spindles or by the pulling apart of the spindle poles
What is telophase?
- Daughter chromosomes arrive at pole and
- nuclear envelope assembles and
- centromeres move apart with contractile ring of actin and myosin formed
- which squeezes the cell so it divides into 2 daughter cells (cleavage furrow)
What occurs in cytokinesis?
Insertion of new membrane at cleavage furrow -> mid-body = where actin-mysoin ring is formed)
What is the spindle assembly checkpoint?
- Checkpoints to ensure everything is in place ->
- between metaphase and anaphase,
- checking whether all the chromosomes are in line and
- for spindles to be assembled ->
- signal is emited when kinetochore isn’t attached to microtubules (like the changing of wheels in formula 1 when the mechanic lets them know that everything is done),
- hoping that all signals are stopped after metaphase
What does the spindle assembly checkpoint require?
2 important proteins in checkpoint -> CENP-E and BUB protein kinase (dissociate from kinetochore when chromatids attached properly to the spindle, then onto signal progression to anaphase
How does misattachment of microtubules occur?
- Normal attachment: one microtubule array’s centrosome is attached to kinetochore of one sister chromatid and microtubule array from other centrosome is attached to kinetochore of another chromatid, allowing sister chromatids to split and move to different poles.
- BUT:
- Syntelic -> both kinetochores hooked by 2 microtubule arrays from SAME centromere;
- Merotelic -> more than one microtubule array attached to same kinetochore (one chromatid pulled in different directions);
- Monotelic -> only one of the kinetochores of one chromatid is attached to microtubule array, the other unattached
How does aberrant centrosome/DNA duplication occur?
If centrosomes not duplicated properly then could end up with 4 in one cell; leading to abnormal attachment of microtubule arrays to kinetochores leading to abnormal cytokinesis
How can you exploit aneuploidy using drugs to make anti-cancer drugs?
- Can slow down cancer by inhibiting proliferation of tumour cells ->
- exploiting checkpoint control by inhibiting the kinetochore signalling to make the nucleus think that it is correctly hooked,
- altering microtubule dynamics, which can cause long term mitotic arrest
How do taxanes and vinca alkaloids work?
Inhibiting the kinetochore signalling to make the nucleus think that it is correctly hooked, altering microtubule dynaiscs, which can cause long term mitotic arres
What occurs to the cell if something goes wrong during the cell cycle?
Cell cycle arrest temporarily or permanently leading to apoptosis
At what points in the cell cycle can tumours form?
- First checkpoint = G1,
- next just before mitosis to check for DNA damage,
- next metaphase-anaphase checkpoint;
- They can also prevent the exit cell cycle G0 and so prevent the dismantlement of the cell cycle apparatus
What are the signalling cascades that occur through the cell?
How does signalling by peptide growth factors work?
What is protein phosphorylation?
How does signalling by peptide growth factors work?
What are the protein kinase cascades and what do they lead to?
x
What is the role of c-Myc?
- Cell cycle entry ->
- conc of Myc is v. low when cell is in G0 but when cell division is triggered (adding GF),
- rapid and dramatic rise in Myc occurs,
- then plateauing at intermediate level,
- moving from G0 to G1.
- Myc is a transcription factor which controls expression of other genes (involved in cell cycle)
What do mitogenic growth factors stimulate in the signalling pathway?
- GF arrives and binds to receptor (tyrosine kinase type) acting via GTP-binding protein (Ras) triggering a kinase cascade.
- Early stage of cell cycle triggering is very fast, which then triggers activation of genes that are required for progression of cells through the cell cycle (slower due to transcription/translation needed)
- c-Myc is triggered in kinase cascade (early),
- which then regulates expression of many other genes
What occurs in signalling by peptide growth factors?
- Phosphorylated receptor protein tyrosine kinase recruits adapter and signalling proteins ->
- dimeric GF bind to RPTK,
- causing cross phosphorylation of tyrosine residues (using gamma phosphate of ATP);
- phosphorylated domains act as docking sites for adaptor proteins which contribute to downstream signalling ->
- herceptin can interfere here;
- Grb2 adaptor molecule is recruited
What are adaptor proteins?
- Grb2 is an important adaptor molecule ->
- has 2 types of protein-protein interaction:
- SH2 (binds to phosphorylated tyrosines of receptor) and
- SH3 (binds to proline rich regions of other proteins)
What is Ras?
- GTP binding protein family -> molecular switches; transient signal.
- Need Sos to enable GTP exchange;
- GAP help Ras shed the phosphate = both help for quick activation and inactivation (not kinases).
- Self-regulating system so Ras can turn itself off (intrinsic GTP hydrolysis capability) and hydrolysis can also be stimulated by GTPase activating proteins ->
- need to be able to turn off the signalling activity of ras to prevent uncontrolled division
How do RPTK’s signal to Ras?
- Grb is bound to RPTK via SH2 and binds to protein called Sos (exchange factor for Ras) via SH3;
- when RPTK is activated receptor is phosphorylated and Grb2 binds to tyrosine domain,
- so Sos is able to get close enough to the membrane to activate Ras (allows exchange of GDP for GTP)
- which changes conformation of Ras, putting it into an active state that can signal downstream and allow for propagation of signal
How can Ras be oncologically activated?
- By mutations that increase the amount of active GTP-loaded Ras.
- V12Ras -> glycine residue in postion 12 changes to valine due to gene mutation, which turns side chain hydrophobic, preventing GAPs from binding to Ras so it can’t turn off very easily.
- L61Ras -> Glutamine in p61 converts to leucine which means side chain goes from amide to hydrophobic, inhibiting intrinsic GTPase activity of Ras protein, so Ras ends up constantly in GTP bound state so gives growth stimulatory signals
What does Ras activate?
Protein kinase cascades -> ERK and MAPK; ERK involved in growth stimulatory signalling
What is the ERK cascade?
ERK specific kinases: Raf -> MEK -> ERK
What do protein kinases stimulate in cell proteins and gene expression?
- Changes -> to promote division;
- At end of ERK cascade, last kinase phosphorylates a number of proteins and changes their activity (such as gene regulatory proteins/TF),
- which then go onto regulate gene expression ->
- c-Myc most important gene turned on by pathway. Ras and Myc are oncogenes as they are most commonly mutated/over-expressed in human tumours
E
What is cell cycle control based on and what are Cdks?
- Cyclically activated protein kinases -
- Cdks= family of protein kinases, serine-threonine kinases;
- not activated until bind to cyclin and are also controlled by phosphorylation
What do cyclins activate and what are cyclins?
Cdks; cyclins are transiently expressed during cell cycle and once activated cdks, they are degraded
Which different events do different cyclin-cdk complexes trigger in the cell cycle?
- M-phase promoting factor controls progression through mitosis,
- cdk activated by mitotic cyclin;
- once cdk’s role is fulfilled,
- cyclin degraded and cdk is turned off
What is mitosis promoting factor?
Cdk1 and mitotic cyclin (usually cyclin B)
How are cyclins expressed?
In a regulated manner; cyclical nature of induction of cyclins controlling mitosis
How are Cdks regulated by phosphorylation and how is MPF activated?
OVERALL: Cyclin binding to Cdk, activating phosphorylation by CAK and removal of inhibitory phosphate placed by Wee1 by Cdc25 all activate MPF.
- Cdk-1 binds to cyclin B, with cdk-cyclin complex usually inactive on its own,
- needing phosphorylation (TWO reactions) to regulate ativity of cdk ->
- cdk needs to be activated at specific sites:
- by Cdk activating kinase, inhibitory kinase Wee1 balances it.
- Cak = activating phosphorylation on Cdk1;
- Wee1 = inhibitory phosphorylation onto cdk1.
- Cdc25 takes off inhib phosphate put on by Wee1;
- so you get active MPF.
How is Cdk1 activated and when?
- Dephosphorylation of inhibitory site of Cdk1 by Cdc25 activates it towards end of interphase,
- active MPF can phosphorylate Cdc25 to increase its activity,
- forming positive feedback that drives mitosis
What is anaphase promoting complex C?
- MPF (active) at end of metaphase,
- phosphorylates key substrates involvd in mitotic process putting mitosis on hold.
- Can’t progress until signal is sent saying metaphase has been correctly achieved;
- once kinetochores have been correctly attached to microtubule spindles,
- signal is released to degrade cyclin B,
- inactivating cdk1 and substrates are dephosphorylated, so mitosis can progress
Which cyclins and Cdks are required at different stages of the cell cycle?
- G1/S -> Cdk2 and cyclin E;
- S -> Cdk2 and cyclin A;
- Mitosis -> Cdk1 and cyclin B;
- same cdk in both G1/S and S but doing different jobs as when cyclin binds to Cdk it actually changes substrate specificity so it can phosphorylate different substrates depending on which cyclins are bound to it
How do growth factors stimulating signalling pathways promote G0 to G1 transition?
- Gf come and bind to RPTK and through ras trigger a kinase cascade,
- leading to phosphorylation of TF that turn on cMyc expression,
- which stimulates transcription of cyclin D
What does Cyclin D do?
Activates Cdk4 and Cdk6 to stimulate synthesis of cyclin E
How is cyclins/cdks expression regulated?
- Gf leads to production of Myc which stimulates synth of cyclin D which leads to production of active cdk4/6-cyclin D complex;
- which then stimulates synthesis of next cyclin and become sequentially active,
- each cyclin involved in stimulating synth of next cyclin ->
- gives direction to cell cycle and
- timing as it takes time for conc of cyclin to build up so appropriate cdk is activated
What do cdks do?
- MPF phosphorylates proteins involved in mitosis (nuclear lamins - which break down nuclear envelope);
- start kinase is a complex of Cdk2 and G1 cyclin which phosphorylates substrates needed for that phase = RETINOBLASTOMA is most important
How do retinoblastomas (RBM) regulate gene expression (up to cyclin E transcription)?
- Present throughout cycle;
- in G0 is unphosph. and it binds to and sequesters E2F (transcription factor family),
- which in cytoplasm unphosph = turned off.
- RBM is target for Cdk4/6-cyclin D kinase (active after Myc induction),
- and starts to phosph RBM protein which oses its affinity for E2F,
- releasing it so it can bind to promoters in nucleus of genes involved in cell cycle progression ->
- RBM acts as brake in cell cycle = TUMOUR SUPPRESSOR GENE
Which genes are regulated by E2F transcription factor?
- Proto-oncogenes including Myc proteins ->
- control genes involved in S phase and cyclin E is one of first targets of E2F,
- pushing forwards next phase of cell cycle
How do RBM’s regulate gene expression after Cyclin E transcription?
- Cyclin E forms complex with Cdk2 and RBM is target for phosph by active Cdk2-cyclin E complex;
- so retinoblastoma becomes further phosph and releases more E2F so conc of E2F in cytoplasm rises
What is the overall activity of cdk?
- Myc turns on cyclin D which complexes with Cdk4/6,
- which phosph RBM and allows E2F to start being released in cytoplasm,
- stimulating further production of cyclin E;
- which forms complex with cdk2 and then complex further phosph RBM leading to further increase in E2F,
- so E2F can now bind to targets with lower affinity,
- which when conc of E2F is high enough activates cyclin A gene promoter and cycle continues throughout the cell
What other regulation of cdks are available and what are the 2 families?
Cdk inhibitors add an extra layer of regulation
What is the importance of the cell cycle regulators in cancer?
Genes commonly lost are tumour suppressor genes; commonly over-expressed are oncogenes
What are the different genes that can be over/underexpressed in cancers and which kind of tumours are they most common in?
- Many tyrosine kinase receptors are oncogenes -> Her2;
- Ras gene is mutationally activated in many cancers and can be targeted by inhibition of membrane attachment
What are the anti-cancer drug targets in the cell?
B
