Carcinogenesis Flashcards
(127 cards)
1
Q
Why are DNA double helixes susceptible to damage?
A
Nitrogenous bases are FLAT, PLANAR, CARBON rings with lots of FUNCTIONAL GROUPS which means that they can easily react with other chemicals e.g. carbon ring makes base susceptible to electron delocalisation. Double bonds between bases are also reactive.
2
Q
What are the different mechanisms of DNA damage? (x4)
A
DEAMINATION: bases are nitrogenous so contain amine groups. Deamination is the removal of an amine group. When an amine group is removed, the amine group is converted into a keto-group (=O group). This results in base changes e.g. CYTOSINE–>URACIL, adenine to hypoxanthine… = BASE MISMATCH MUTATION, creates a bulge in DNA structure.
CHEMICAL MODIFICATION: double bonds open and oxidation reactions subsequently occur at bases. Opening of the double bond is caused by hyper-reactive oxygen species which are generated from normal metabolism or ionising radiation. COMMON CHEMICAL MODIFICATION: double bond opens, and thymine base becomes thymine glycol with two -OH groups (see photo). Things can be added to the -OH groups. If these chemical species are large, they create ADDUCTS (describes large molecule covalently bonded to DNA) which can be carcinogenic.
PHOTODAMAGE: damage from UV light. Occurs WITHIN a strand rather than between strands where there two adjacent thymine bases: UV light activates the thymine bases, which become reactive and form THYMINE DIMERS – distorts double helix.
RADIATION: can break phosphodiester bonds which form the DNA backbone. When a bond breaks, it creates NICKS in the DNA. When there are lots of nicks, whole GAPS can form where a whole region of single strand is missing.
3
Q
What are the different types of DNA damage? (x5)
A
- Base dimers.
- DNA adducts and alkylation (addition of CnH2n+1 groups).
- Formation of abastic bases (base is destroyed so much that it is no longer a base) aka apurinic or apyrimidinic sites.
- Base mismatches from base changes.
- Single and double DNA strand breaks (from nicks and gaps).
4
Q
What are the causes of DNA damage? (x2)
A
• CHEMICALS that causes cancer – CARCINOGENS e.g. dietary, lifestyle, environmental, occupational, medical and endogenous. • RADIATION e.g. ionizing, solar and cosmic.
5
Q
How does DNA damage lead to cancer?
A
DNA damage lead to mutation which can lead to cancer.
6
Q
How can DNA damage be used in medicine?
A
DNA damage can be utilised in chemotherapy.
7
Q
What type of compounds are carcinogens?
A
Polycyclic Aromatic Hydrocarbons with double bonds. They can also have substituents e.g. methyl groups and N or S inside rings.
8
Q
How are carcinogens processed by the body?
A
• They are treated in the same way as drugs in drug metabolism. When they are metabolised, this process is called CARCINOGENESIS. • PHASE I METABOLISM: addition of functional groups to make carcinogen more reactive – through oxidation, reduction or hydrolysis. Mainly CYTOCHROME p450-mediated. • PHASE II METABOLISM: conjugation of Phase I functional groups (through sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation) to produce POLAR (water soluble) metabolites for excretion.
9
Q
How does metabolism of Benzo[a]pyrene lead to mutagenesis?
A
- B[a]P is not itself carcinogenic. Its metabolites are.
- B[a]P is metabolised by cytochrome p450s and Epoxide-Hydroxylase in the liver.
- Result: forms an EPOXIDE form of B[a]P which is highly carcinogenic.
- This molecule attaches itself to chemically reactive regions of DNA, forming ADDUCTS –> mutations and cancer.
10
Q
How does the metabolism of Aflatoxin B1 lead to mutagenesis?
A
• Aflatoxin B1 is formed by mould and found on grains and peanuts. • It is a potent liver carcinogen. • Cytochrome p450 converts it into an epoxide. • This epoxide-form of Aflatoxin B1 reacts with DNA (specifically guanines in the N7 position) and results in adduct formation –> leading to mutation.
11
Q
How does the metabolism of 2-naphthylamine lead to mutagenesis?
A
• From dyes and a potent bladder carcinogen. • In Phase I metabolism, the cytochrome p450 tries to detoxify 2-napthylamine. • In Phase II metabolism, glucuronide is added to the chemical by glucuronyl transferase. • When it is excreted, urine pH in the bladder causes this glucuronide formation to break down and produce an electrophile which can react with DNA –> MUTAGENESIS.
12
Q
How do oxygen free radicals cause DNA damage? (x3)
A
• Oxygen free radicals possess unpaired electrons, are electrophilic, and therefore seek out electron-rich DNA. • It creates APURINIC and APYRIMIDINIC sites (abastic bases i.e. destroyed). • Oxygen free radicals do this by (i) opening guanine and adenine rings, (ii) creating thymine and cytosine glycols, (iii) convert purines into 8-hydroxypurines (A and G) – more chemically reactive so can undergo adduct formation.
13
Q
What is the normal state of endogenous DNA repair and damage? However, note of caution?
A
DNA is damaged all the time inside the body. However, the max DNA repair rate FAR EXCEEDS the rate of damage. Therefore, human cells have plenty of spare capacity to deal with both endogenous and exogenous damage. HOWEVER, errors creep in especially with increasing age.
14
Q
What is genetic underpinning of DNA repair pathways?
A
The tumour suppressor gene, p53 is responsible for activation of repair pathways. It is a transcription factor and responsible for regulation of gene involved in these pathways. Normally, p53 is held in an inactive form by MDM2. When DNA damage occurs, the MDM2 is lost and p53 is activated.
15
Q
What are the four different types of DNA repair?
A
• DIRECT REVERSAL OF DNA DAMAGE. • BASE EXCISION REPAIR: used mainly to repair sites where the base has been lost i.e. sites of apurinic or apyrimidinic damage. • NUCLEOTIDE EXCISION REPIAR: mainly repair DNA where there are adducts. • DURING OR POST-REPLICATION REPAIR includes MISMATCH REPAIR and RECOMBINATIONAL REPAIR.
16
Q
What is the mechanism of direct reversal of DNA damage (or direct DNA repair)?
A
• Involves the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions. • PHOTOLYASES repair pyrimidine-DIMERS (including thymine dimers). • O6 METHYLGUANINE-DNA METHYLTRANSFERASES (MGMT) and ALKYLTRANSFERASES remove alkyl groups from bases i.e. reverses simple alkylation adducts.
17
Q
What are alkyl groups?
A
Contains only carbon and hydrogen atoms: CnH2n-1. Most commonly, these are METHYL (CH3) groups.
18
Q
What is the mechanism of DNA mismatch repair?
A
- Repair mismatches rather than mutations e.g. DNA polymerase has put in the wrong base during DNA replication.
- The ‘bulge’ produced by a mismatch is recognised by MSH proteins, and MLH protein and a nuclease cuts out the offending base and surrounding regions of the strand.
- DNA Polymerase restores the removed region of DNA.
- This mechanism only works during DNA replication!
19
Q
What is the mechanism of base excision repair of DNA damage?
A
- Repair sites where the base has been lost i.e. sites of apurinic or apyrimidinic damage.
- DNA GLYCOSYLASES remove the base part of DNA without affecting the phosphodiester bonds of the backbone.
- AP-ENDONUCLEASE cuts the DNA backbone where the base has been removed.
- DNA POLYMERASE fills in the strand with the correct and functional base.
- DNA LIGASE repairs the phosphodiester backbone.
20
Q
What is the mechanism of nucleotide excision repair of DNA damage?
A
- Repair DNA where there are big adducts.
- XERODERMA PIGMENTOSUM proteins recognise these sites of DNA damage.
- TRANSCRPTION FACTOR II H (TFHII) and some xeroderma pigmentosum proteins unwind the DNA (have helicase activity). They also have ENDONUCLEASE activity, forming nicks around the mutated base.
- A patch of DNA is removed by these proteins across the strand where nicks were inserted.
- DNA POLYMERASE fills in the gap.
- DNA LIGASE repairs the phosphodiester backbone.
- Very similar to base excision repair, but a larger operation.
21
Q
What is transcription-coupled nucleotide-excision repair (NER)?
A
Most of the genome undergoes NER when the DNA is silent i.e. not doing anything. However, NER can also occur when the DNA is ACTIVE and being transcribed – this is called transcription-coupled nucleotide-excision repair (TC-NER). TC-NER differs from normal NER only in being FASTER and having different INITIAL STEPS. TC-NER does not require xeroderma pigmentosum to recognise damage, but NER is instead initiated when RNA polymerase stalls at areas of damage when transcribing the DNA strand.
22
Q
What genetic diseases involve nucleotide-excision repair? (x3)
A
Xeroderma Pigmentosum, Trichothiodystrophy and Cockayne’s Syndrome.
23
Q
What is the mechanism of the disease Xeroderma pigmentosum?
A
Xeroderma pigmentosum proteins are important in the recognition and removal of damaged regions of DNA. XP is a rare inherited disease of mutations of these proteins.
24
Q
What are the symptoms of Xeroderma pigmentosum?
A
Severe sensitivity to UV light which manifests itself by the formation of skin cancers. In such patients, there is severe pigmentation irregularities, elevated frequency of other forms of cancer and frequent neurological defects.
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What is Trichothiodystrophy?
Sulphur deficient brittle hair, facial abnormalities, short stature, ichthyosis (fish-like scaly skin) and light sensitivity, from mutations in the genes responsible for transcription-coupled nucleotide-excision repair.
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What is Cockayne’s syndrome?
Dwarfism, light sensitivity, facial and limb abnormalities and neurological abnormalities from mutations in the genes responsible for transcription-coupled nucleotide-excision repair.
27
What are the mechanisms of recombinational repair? (x2)
* Occur when damage to DNA is substantial – when DNA DOUBLE STRAND is broken. There are two mechanisms:
* DIRECT JOINING: When double strand breaks, the ends do not match, so exonucleases cut back the ends until they reach sequences of DNA that are complementary to the DNA of the other strand. Once these have been located, the two strands are brought back together again by normal base pairing (of course though, the exonucleases mean that some DNA is now missing). DNA polymerases clear up any gaps and ligases fix the backbone – see photo.
* NONHOMOLOGOUS END-JOINING: when complementary nucleotides are not found/searched for, a protein called Ku holds the two ends together and forces them to join back.
28
What are the consequences of DNA damage? (x3)
• EFFICIENT REPAIR leading to a normal cell. • INCORRECT REPAIR leading to an altered primary sequence. This will create FIXED MUTATIONS in the cell in the DNA that are carried through replication and cell division. • APOPTOSIS if damage is very substantial.
29
What are the possible outcomes of fixed mutations? (x2)
Transcription/translation giving aberrant proteins OR carcinogenesis if critical targets are mutated: activation of oncogenes and inactivation of tumour suppressor genes.
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What does aberrant mean?
Diverging from the normal type.
31
How can we test for carcinogenicity of chemicals? (x4)
The following tests are SEQUENTIAL. If the chemical is positive in the first test, it is tested using the second test. Each time, the tests give a more comprehensive assessment of CARCINOGENICITY.
1. AMES TEST: in vitro BACTERIAL gene mutation assay – chemical to be tested is mixed with a rat liver enzyme preparation (called S9 preparation – containing p450c and enzymes for Phase I and II metabolism) AND bacteria with a mutation that means it cannot synthesise histidine. If bacteria mutate so that it acquires the ability to synthesis histidine, it indicates that the chemical is MUTAGENIC and therefore may act as a carcinogen. Histidine is needed for growth, so histidine synthesis is indicated by a colonised culture plate.
2. CHROMOSOMAL DAMAGE TEST: in vitro MAMMALIAN CELL assay – mammalian cells are treated with chemical in presence of liver S9 preparation. Look for damage microscopically e.g. chromosome exchange, acentric rings, chromosome breaks and gaps.
3. MICRONUCLEUS ASSAY: in vitro micronucleus assay – cells treated with chemicals and allowed to divide. If DNA becomes very damaged, it gets budded off into MICRONUCLEI during cell division – presence of these can be studies by microscopy.
4. MURINE BONE MARROW MICRONUCLEUS ASSAY: IN VIVO mammalian assay – treat animals (usually rats) with the chemical we want to test and examine bone marrow cells in peripheral blood erythrocytes for micronuclei.
32
What is angiogenesis?
The formation of new blood vessels.
33
What are the physiological causes of angiogenesis? (x3)
Development, menstrual cycle and wound healing.
34
What are the main pathological causes of angiogenesis? (x4)
Cancer, chronic inflammatory diseases, retinopathies, ischaemic diseases and more.
35
What is vasculogenesis?
Angiogenesis is not the only way to make blood vessels. Vasculogenesis produces new vasculature from terminal vessels from differentiation of bone marrow precursor cells into endothelial cells.
36
What is arteriogenesis?
Angiogenesis is not the only way to make blood vessels. Arteriogenesis refers to an increase in the diameter of existing arterial vessels.
37
What are angiogenesis regulators?
• The angiogenic process is regulated by a wide array of growth factors and signalling pathways. • Most of these pathways depend on the dynamic regulation of gene expression in endothelial cells and are determined by a complex network of transcriptional regulators. • There are angiogenesis ACTIVATORS, INHIBITORS and those involved in maintaining the INTEGRITY of newly formed vessels. Some activators are essential e.g. VEGF; others are ideal but not a requirement. Some are both pro- and anti-angiogenic.
38
What are the basic steps of angiogenesis?
1. SELECTION OF SPROUTING ENDOTHELIAL CELLS: a TIP cell is activated, as well as the cells sitting below it. The cells adjacent to the tip cell are STALK cells. There is lateral growth inhibition.
2. SPROUT OUTGROWTH AND GUIDANCE: Tip cells navigate the direction of angiogenesis, while stalk cells proliferate – resulting in SPROUTING of the endothelium.
3. Branching coordination.
4. SPROUT FUSION AND LUMEN FORMATION: Stalk elongation and tip cell fusion (anastomosis). Lumen formation also occurs in this part.
5. PERFUSION AND VESSEL MATURATION.
39
What are physiological triggers for angiogenesis?
Hypoxia.
40
How does hypoxia induce angiogenesis?
• Hypoxia-inducible transcription factor (HIF), in presence of oxygen, is inhibited by pVHL (Von Hippel-Lindau tumour suppressor gene protein). pVHL binds to HIF, preventing it from exercising its effects. • When there is hypoxia, HIF detaches from pVHL and binds to DNA. It is a transcription factor which promotes the expression of genes involved in angiogenesis including VEGF.
41
What are the types of VEGF and its receptors?
There are 5 members in the VEGF family: VEGF A-D, and placental growth factor (PlGF). VEGF can bind to three TYROSINE KINASE RECEPTORS (VEGFR-1, 2 and 3) and two neuropilin co-receptors (Nrp1 and 2).
42
What happens in selection of sprouting endothelial cells in angiogenesis?
1. Something in the tissue environment produces VEGF.
2. VEGF binds to a VEGF receptor on an endothelial cell.
3. The cell that VEGF binds to becomes the tip cell. In activating a tip cell, the adjacent endothelial cells become stalk cells which proliferate to support sprout elongation. This is mediated by NOTCH SIGNALLING.
4. The basement membrane degrades at the area of sprouting, and there is pericyte detachment and loosening of endothelial cell junctions.
5. Subsequent increased permeability of blood vessel wall in this region permits extravasation of plasma proteins (such as fibrinogen and fibronectin) to deposit a provisional matrix layer, and proteases remodel pre-existing interstitial matrix, all enabling cell migration.
43
What happens in Notch signalling?
1. VEGF binds to tip cell, resulting in upregulation of the notch ligand, DLL4. 2. DLL4 binds to notch receptors on adjacent endothelial cells, driving notch signalling in those cells. 3. In notch signalling, the intracellular domain of notch receptors in adjacent endothelial cells CLEAVES itself from the receptor – called intracellular domain of notch (NICD). NICD translocates to the nucleus and binds to transcription factor RBP-J. 4. This Notch signalling inhibits the expression of VEGFR2 (receptors) in stalk cells. 5. RESULT: tip cells acquire a sprouting phenotype; adjacent cells become stalk cells and proliferate to support sprout elongation.
44
What happens in sprout outgrowth and guidance?
* Tip cells navigate in response to guidance signals (such as semaphorins and ephrins).
* Tip cells also adhere to surrounding extracellular matrix (mediated by integrins).
* Stalk cells behind the tip cell proliferate, elongate and form a lumen.
* Proliferating stalk cells attract pericytes and deposit basement membranes to become stabilized.
* Myeloid cells (such as macrophages) support sprouting – macrophages help by carving out tunnels in the extra cellular matrix (ECM), providing avenues for capillary infiltration by the sprout. Macrophages also proteolytically liberate angiogenic growth factors embedded in the ECM as it carves through it, and are involved in anastomosis at tips of sprouting vessels.
45
What is the role of platelets in angiogenesis?
Platelets are full of regulators of angiogenesis. Their overall role is to MODULATE angiogenesis – they contain both pro- and anti-angiogenic mediators.
46
What happens in perfusion and vessel maturation in angiogenesis? (x2)
• After fusion of neighbouring branches, lumen formation allows perfusion of the neovessel. • The new vessel is stabilised in many ways: • FORMATION OF TIGHT AND ADHEREN JUNCTIONS between endothelial cells – by mediators. Junctions are formed between hydrophilic transmembrane proteins. Transmembrane means that endothelial cells can communicate with each other. • MURAL CELLS (PERICYTES) FORM: around the neovessels and are crucial for membrane stability by producing molecules such as in the ANGIOPOIETIN-TIE-2 SYSTEM.
47
What is a neovessel?
A new blood vessel.
48
What is the importance of the formation of junctions between endothelial cells? (x5)
• Allows for communication between endothelial cells. • Mediates adhesion between endothelial cells. • Fundamental to permeability of blood vessels and the ability of inflammatory cells to enter tissues. • Control CONTACT-INHIBITION of cell growth. • Promote survival of endothelial cells.
49
What is contact inhibition?
Contact inhibition refers to two different processes: CONTACT INHIBITION OF LOCOMOTION (or cell growth) – when two cells contact each other, they attempt to alter their locomotion in a different direction to avoid future collision (this occurs in endothelial cells to keep endothelium one-cell thick); and CONTACT INHIBITION OF PROLIFERATION – when cells stop proliferating and growing if they cannot prevent collision by changing direction.
50
What is the angiopoietin-Tie-2 system?
• This system is important in the regulation of endothelial activation (--\> angiogenesis). • Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are antagonistic ligands, that bind to the extracellular domain of the Tie-2 receptor on endothelial cells. • Ang-1 promotes VESSEL STABILITY, inhibits vascular leakage and suppresses inflammatory gene expression. It is produced by PERICYTES. • Ang-2 is stored in Weibel–Palade bodies of endothelial cells. It antagonises Ang-1 and has similar actions to VEGF – promotes vascular instability and VEGF-dependent angiogenesis (explained earlier).
51
What is the relationship between tumour growth and angiogenesis? What links them together?
Up to a certain size, tumours can grow without new vasculature. However, above a certain size, ANGIOGENESIS is needed to ensure exponential tumour growth. The point that angiogenesis is needed for tumour growth is called the ANGIOGENIC SWITCH.
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What does the angiogenic switch depend on?
The angiogenic switch can occur at different points in the tumour-progression pathway depending on the nature of the tumour and its microenvironment.
53
How are tumour blood vessels abnormal? (x5)
• Irregular shaped, dilated, tortuous (twisting). • Not organised into definitive venules, arterioles and capillaries. • Leaky and haemorrhagic, partly due to the overproduction of VEGF and infiltration by inflammatory mediators. • Perivascular cells often become loosely associated. • Some tumours may recruit endothelial progenitor cells from the bone marrow.
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How do tumours promote angiogenesis? (x3)
• Cancer cells contain fibroblast cells which differentiate into CANCER-ASSOCIATED FIBROBLASTS (CAFs). These secrete ECM and pro-angiogenic growth factors including VEGF-A and FGF2. • PERICYTES are loosely attached with the tumour-associated blood vessels which favours chronic leakage of tumours. This is enhanced by angiopoietin 2 which is pro-angiogenic. • Tumours promote PLATELETS which release predominantly pro-angiogenic mediators and proteases that support the proliferation of CAFs. This also promotes platelet aggregation.
55
What therapies inhibit VEGF signalling? (x2)
• THERAPIES THAT INHIBIT VEGF, INHIBIT ANGIOGENESIS. • VEGF inhibition by soluble VEGFR1: VEGFR-1 (a VEGF receptor) ‘mops up’ VEGF preventing it from stimulating angiogenesis. • AVASTIN: an anti-VEGF (antibody) which stops VEGF before it can bind to VEGFRs and promote angiogenesis.
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What is the problem with inhibiting VEGF in cancer therapy? (x2 +1)
• There are lots of side-effects because VEGF is not just a pro-angiogenic factor, but is also essential for endothelial cell survival (which effects all endothelial cells in body). • More specifically, anti-VEGF therapies may damage healthy vasculature leading to loss of vessels, creating vasculature resistant to further treatment and inadequate for delivery of oxygen/drugs. • Tumour becomes resistance to inhibition of VEGF.
57
What are the possible mechanisms of resistance to anti-VEGF therapy in cancer? (x4)
1. By making tumour hypoxic, tumour produces another angiogenic factor (growth factor).
2. Tumours vessels may be less sensitive to VEGF inhibition due to vessel lining by tumour cells or endothelial cells derived from tumours.
3. Tumour cells that recruit pericytes may be less responsive to VEGF therapy.
4. Vasculogenic mimicry – tumours pretend to be vessels and form their own vessel-like channels within the tumour. Vessels hook up to the channels, and the channels perfuse the tumour mass without vessels having to sprout carve their own way (see photo).
58
What strategy overcomes the issues of anti-VEGF therapy? Result? (x2)
• Anti-VEGF therapy damages healthy vasculature leading to loss of vessels, comes with many side-effects and tumours may become resistant. • Therefore, an alternative strategy is to NORMALISE THE VASCULATURE OF TUMOUR CELLS i.e. make them less abnormal, and more like normal vessels in the body. • Therefore, hypoxia-induced angiogenesis will be reduced, preventing further neovessel formation. • Normalising vessels in the tumour also INCREASES the efficacy of conventional treatments such as chemotherapy which uses blood to access tumours.
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What are the laboratory challenges of developing therapeutic strategies to inhibit angiogenesis in cancer?
• Tumours are complex three-dimensional (3D) structures with their own unique microenvironments • We lack good in vitro models - our understanding of tumour behaviour in a complex 3D environment is limited and drug screens are often misleading. • Studies are performed on cell lines growing as two-dimensional (2D) monolayers, which do not mimic the complex interplay between tumour cells and their extracellular environment • The phenotype of tumour cells when cultured in 2D vs 3D is different • Crucially, tumours receive nutrients and therapeutics through the vasculature, which is not included in any in vitro tumour models. • This is a big challenge to developing therapeutic strategies to inhibit angiogenesis in cancer.
60
What are the stages of tumour progression to local invasion?
1. There is normal homeostasis. In the case of epithelium, the epithelial cells are tightly cohesive, polarised (distinct apical and basal membranes), and separated from the stroma by a basement membrane. 2. Genetic alterations lead to hyper-proliferation. 3. Accumulation of mutations leads to differentiation: there is disassembly of cell-cell contacts and cells lost cohesion with each other. Cells – in the case of epithelium – also lose their polarity. 4. Cells form a mass and invade the basement membrane (it’s not a malignant tumour). There is increased motility, and cells cleave the ECM proteins of the tissue stroma – carving channels through the tissue to enable local invasion.
61
What happens during local invasion that allows for cancer cells to metastasise? How do the cells change? What happens once the cells have metastasised?
Cells acquire mobile mesenchyme-type cell phenotype and able to migrate more easily. They enter the blood stream. Cells travel through the blood stream and exit the circulation to invade new organs. On invasion, they lose their mesenchymal characteristics to form a new tumour.
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What is mesenchyme? Compared to epithelial cells?
Mesenchyme is an embryonic tissue that develops into cells of the lymphatic and circulatory systems and of connective tissues. The cells are loosely organised and characterised by ECM and an ability to migrate easily – in contract to epithelial cells which lack mobility, are highly cohesive and polarised.
63
What are the different types of tumour cell migration? (x4)
• TRAVEL AS SINGLE CELLS: • AMEOBOID – migrate as round structures. • MESENCHYMAL – single cells with elongated structure. • CELLS TRAVEL IN CLUSTERS/cohorts. • CELLS TRAVEL IN MULTICELLULAR STRANDS/SHEETS.
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What is the difference in metastatic potential of different types of tumour cell migration?
Clusters have higher metastatic potential than single cells.
65
What are the molecules required during tumour cell migration for motility? (x5)
• In single cell and multicellular migration (clusters and strands), INTEGRINS and PROTEASES are both required. Integrins are found on cells and recognise ECM proteins – they facilitate mobility of the cancer cells. Proteases are important for breakdown of surrounding ECM for migration. • In multicellular migration (but not in single cell migration), CADHERINS and GAP JUNCTIONS are important for cell-cell coordination within clusters and strands. Cadherins (found on epithelial cells) induce differentiation, and gap junctions facilitate cell-cell communication and promotes invasion. • ACTIN FILAMENTS and their different arrangements are responsible for the physical movement of cells.
66
Biomimicry in metastasise?
Cancer metastasises mimic normal morphogenic events e.g. angiogenesis, branching morphogenesis (in mammary gland) and ovarian egg cells. In these normal morphogenic events, there is a leading cell, or leading cluster of cells that direct the growth/migration. Similarly, in tumours, there are TIP CELLS which coordinate migration.
67
What happens to gene expression that facilitates metastasise?
There is upregulation of genes involved in CYTOSKELETON REGULATION and MOTILITY MACHINERY.
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What stimuli promote movement of cells?
Organogenesis, morphogenesis, wounding (cells move to close gaps created by wounds), growth factors, chemo-attractants (WBC migration) and DEDIFFERENTIATION (in tumours).
69
How do cells move?
1. Cell makes focal adhesions (adhesions with the substratum).
2. Cell EXTENDS and forms a new adhesion further along in its direction of movement.
3. There is cellular contraction and TRANSLOCATION of the cell cytoplasm towards the new adhesion.
4. De-adhesion of the old adhesion occurs. Think of it like rock-climbing!
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What is the substratum?
An ECM surface.
71
What types of control are needed in cell movement? (x3)
• Control is needed WITHIN A CELL to coordinate what is happening at different parts (or cell rips apart from multidirectional movement). • Control is needed to regulate adhesion/release of cell-ECM receptor to facilitate movement. • Control is needed from outside so that cells can respond to external influences e.g. signals that promote growth such as growth factors, or sensors that tell cell to stop migrating (cell contact inhibition).
72
What are the two types of motility?
Hapoptatic (no purpose); chemotactic (purpose i.e. there is a stimulus).
73
What is the structure of focal adhesions? How do they work?
Focal adhesions are mediated by integrins which are transmembrane proteins on the surface of cells. They sense surrounding substratum (ECM). When they interact with surrounding ECM, a ‘plaque’ of cytoskeleton proteins assembles around the intracellular integrin domains. Each protein interacts with the cell cytoskeleton and mediate cell remodelling of the cytoskeleton to move the cell in the direction of the focal adhesion.
74
What are the two states of actin?
There are two states of actin – monomers (small soluble subunits called G-ACTIN) and large filamentous polymers (called F-ACTIN). The G-actin can quickly develop into F-actin – F-actin is the contractile actin filaments which allow for cellular movement.
75
Polarity of actin within cells?
When a cell is moving in a certain direction, actin with polymerise at the side of the cell that is leading the direction of movement. In other words, filamentous actin is assembled at the leading pole of the cell – there is ACTIN FILAMENT POLARITY.
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What actin filament structures of the cytoskeleton are important for cell movement? What are their structures? Role?
* FILOPDIA – protrusions covering the surface of the cell and rick in PARALLEL actin filaments. They allow migration, sensing and cell-cell interactions.
* LAMELLIPODIA – sheet-like protrusions (lamellae) covering the cell surface and rich in BRANCHED and CROSS-LINKED actin filaments. They are responsible for movement of cell across substratum surface.
* STRESS FIBRES – large bundles of ANTIPARALLEL actin filaments that form thick fibres within the cell which contract and exert the force of the cell movement. Antiparallel arrangement allows this contraction.
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What are the processed involved in actin filament remodelling (that allow for cellular motility)? (x7)
1. NUCLEATION: formation of trimers to initiate polymerisation. It is the LIMITING STEP in actin dynamics. 2. ELONGATION: sequestering (floating around) actin monomers are used for this. 3. CAPPING: prevents further elongation. 4. SEVERING: the break down of the actin filament into G-actins and shorter filaments. 5. CROSS-LINKING and BUNDLING: of actin filaments – forming the structures required for cell motility e.g. Filopodia. 6. BRANCHING: forming branches which are all 70 degrees. 7. GEL-SOL TRANSITION: occurs by actin severing and makes cell more malleable – permitting protrusion and movement.
78
What happens in actin nucleation?
Trimerization is required for polymerisation to occur. However, an actin trimer is unstable. Therefore, Arp2 and Arp3 (which resemble actin monomers) form the ARP complex. A G-actin monomer joins the ARP complex to form a trimer – called a nucleated actin filament. The ARP complex is the MINUS end, and polymerisation can now occur.
79
What promotes and inhibits elongation of F-actin?
Elongation requires PROFILIN (forming ACTIN PROFILIN COMPLEX) – brings together actin. This process is blocked by THYMOSIN (forming actin thymosin complex) which is abundant in cells and binds to G-actin monomers and prevents elongation.
80
What happens in actin filament capping? Examples of capping proteins?
Capping proteins stop actin filament elongation. CAP Z, Gelsolin and Fragmin/Severin cap the plus end of the filament; Tropomodulin and ARP complex cap the MINUS end of the filament.
81
What are the ‘plus’ and ‘minus’ ends of actin filament?
The ‘plus’ end is where G-actin monomers are added (also known as BARBED end); The ‘minus’ end is where G-actin monomers are severed.
82
What proteins are involved in severing? (x3)
Gelsolin, ADF (cofilin) and fragmin (Severin).
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What are the four fates of severed actin filaments?
* DEPOLYMERISATION AND MONOMER RECYCLING: once the monomer is depolarised, ATP is converted into ADP and the G-actin is ready for polymerisation again.
* CAPPING: the BARBED (+) end is capped, preventing elongation.
* ANNEALING: fragment is added to another actin filament.
* GROWTH FROM PRE-EXISTING END: continues growing.
84
What proteins are involved in cross-linking and bundling of actin filaments? (x6)
* Fascin cross-links and bundles actin filament.
* Fimbrin has different spacing between bundled filaments.
* Alpha-actinin is a dimer which cross-links the filaments.
* Spectrin cross-link the actin filaments which allow for mesh-formation of the actin filaments.
* Filamin does the same.
* Dystrophin links filaments to the plasma membrane.
85
What happens in branching of actin filaments?
Held at 70 degrees by ARP complexes (note that these are the same complexes in nucleation).
86
What are bundling proteins?
Hold actin in parallel bundles e.g. Fascin and Fimbrin.
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What are cross-linking proteins?
Hold actin in a gel-like mesh work in the cell cortex (inner face of cell membrane) e.g. Fascin and Fimbrin.
88
What happens in gel-sol transition?
Cross-linking of actin filaments holds the cell in a gel-like mesh work that that is RIGID. When the cell moves, the cytosol and membrane need to be less rigid, so actin filaments severing occurs to break up the mesh arrangement – cell becomes SOL.
89
How do the processes involved in actin remodelling cooperate? Including myosin involvement? !!!
* Filaments are formed, elongated and severed simultaneously. Hence, as actin filaments are polymerised at the + end, they are being broken at the ‘-‘ end also!
* Capping of ends, cross-linking of actin/myosin filaments and formation of branches also occurs simultaneously.
* Where bundling proteins are present between actin filaments, there is NO SEVERING.
* When strands are arranged in opposite directions, strands can slide over each other, allowing for cells to contract (stress fibres – antiparallel actin filaments). Myosin mediates the sliding movement.
* Buckling occurs when contraction converges two fibres together.
* Tension is created by the contractile actions of myosin.
* OVERALL, the processes are DYNAMIC!
90
What actin remodelling processes occur at lamellae protrusion formation (lamellipodia)?
Branching, capping and polymerisation occur at lamellae and promote movement of membrane forward. At the rear, there is simultaneous disassembly of ARP complex (by cofilin) and subsequent severing of the minus end. G-actins migrate to the leading edge of the cell for assembly.
91
How do Filopodia form?
• There is actin assembly at the tip of the filopodia, and bundling proteins bundle the filaments. At the same time, actin filaments are severed at the ‘minus’ end (at the base of the filopodia) – this is known as RETROGRADE FLOW. G-actins and short filaments are brought to the tip for annealing and elongation. • Initially, elongation occurs faster than the severing, hence there is growth of filopodia. • Filopodia stop growing when a capping protein is added to the tip. • The constantly disassembling at the base means that the filopodia retract to the membrane after not long.
92
What are the signalling mechanisms that regulate the actin cytoskeleton? (x4)
1. ION FLUX CHANGES: different levels of intracellular calcium which activate different proteins over others. 2. PHOSPHOINOSITIDE SIGNALLING: many cytoskeletal proteins bind to phospholipids – when this occurs, their properties can change. 3. KINASES/PHOSPHATASES: phosphorylation of cytoskeletal proteins (through kinase and phosphatase cascades) leads to regulation of cytoskeletal structures. 4. SIGNALLING CASCADES VIA small GTPases.
93
How is the actin cytoskeleton controlled? (x3) Activation? (x3)
* Small GTPases control cytoskeleton processes – they are a group of proteins belonging to the Ras family (oncoprotein).
* Small GTPases include, Rho, Rac and Cdc42.
* The small GTPases are activated by addition of GTP. Cleavage of GTP to GDP leads to inactivation – function is therefore very transient. These proteins are activated by receptor tyrosine kinase, adhesion receptors and signal transduction pathways.
* Rho: leads to formation of stress fibres.
* Rac: leads to formation of lamella. If the SITE of expression of Rac is not regulated, then lamellae grow everywhere and cell spreads like a pancake.
* Cdc42: leads to formation of filopodia.
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How do Rac and Cdc42 regulate actin remodelling?
• They regulate actin-binding proteins. • Rac-GTPases regulates WAVE protein which partners with Arp2/3 complex and helps polymerisation AND branching. • Both GTPases regulate Cofilin and Profilin which are involved in severing and elongation respectively. • Cdc42 regulates WASP protein which influence polymerisation.
95
REVISIT: ‘How do cells move?’ – what points do the small GTPases act on cell? !!!
* Rac – involved in formation of lamellipodium through actin polymerisation and branching.
* Rac and Rho – focal adhesion formation.
* Rho – contraction to move cell forward at the rear by assembling stress fibres, controls the myosin motor which creates tension, and mediates contraction.
* Rho – deadhesion.
* Cdc42 – filopodia formation, polarized motility, and actin polymerisation.
96
How is sequestering promoted? (x2)
Sequestering is promoted by thymosin (elongation) and cofilin (clipping ARP complex).
97
What is cell behaviour?
Describes the ways cells interact with their external environment and their reactions to this, particularly proliferative and motile responses of cells.
98
What types of external influences are detected by cells? (x2)
• CHEMICAL – hormones, growth factors, ion concentration, ECM and nutrients. • PHYSICAL – mechanical stresses, temperature, the topography (or layout) of the ECM and other cells.
99
What external factors are important in relation to proliferation of cancer cells? (x3)
Growth factors, cell-cell adhesion and cell-ECM adhesion.
100
What do cells do when the adhere to ECM surface?
They settle on the surface, spread, and acquire motility – they form a lamellipodium which is the leading edge of the cell.
101
What is the importance of cell spreading on ECM? Term to describe this?
When cells spread on ECM surface, they are prompted to enter S-phase and PROLIFERATE. When cells cannot spread, they do not proliferate, even in the presence of growth factors. When cells are not adhesive to ECM, cells do not significantly synthesise protein or DNA. Spreading and attachment to ECM is required to begin protein synthesis and proliferation – ANCHORADE DEPENDENCE.
102
What’s the difference in cellular response when there is cell-ECM adhesion and cell-cell adhesion?
When a cell adheres to an ECM surface, there is spread of the cell, loss of blebbing, and development of a lamellipodium. When there is cell-cell adhesion, the cells do not spread – instead, they BLEB.
103
How does ECM composition affect cell proliferation? What does this tell us about cellular interaction with ECM?
Epithelial cells require the correct ECM matrix composition to differentiate and express polarity and organisation – it has a profound impact on differentiation e.g. see photo – basal lamina is essential for organisation of mammary epithelial cells and phenotype. WHAT DOES THIS TELL US? The effects that matrix-binding can have on cell function suggests that cells can sense the composition of their environment.
104
How are cells able to sense the composition of their environment?
Cells have ECM-binding receptors – INTEGRINS are the most important – which are linked to their cytoplasmic domains – these domains interact with the cytoskeleton. This arrangement means that there is mechanical continuity between ECM and the cell interior.
105
What is the structure of integrins – in relation to their function?
They are not a protein, they are protein heterodimer COMPLEXES of alpha and beta subunits that associate extracellularly by their ‘head’ regions. Each of the ‘leg’ regions spans the plasma membrane. Ligand-binding occurs at the junction of the head regions. There are small cytoplasmic tails.
106
How do types of integrin differ?
There are more than 20 combinations of alpha/beta known – each bind to a specific short peptide sequence on ECM proteins e.g. alpha5-beta1 fibronectin receptor binds to arg-gly-asp amino acid sequence (RGD). Peptide sequences such as RGD are found in more than one EGM molecule e.g. RGD found in fibronectin, vitronectin and fibrinogen.
107
What is the function of integrins? (x2)
Sense the ECM around them (triggering different responses depending on composition) AND sense mechanical properties of their surroundings – both functions arise from adhesions formed w/ ECM.
108
How are integrins able to trigger intracellular responses? Called?
Integrins recruit cytoplasmic proteins (forming a plaque) which promote SIGNALLING and ACTIN ASSEMBLY (the proteins in this case are called actin-binding proteins) when integrins bind to ECM ligands – this is called OUTSIDE-INSIDE SIGNALLING.
109
How do integrins form adhesions? Purpose?
Integrin complexes cluster to form focal adhesions (between cell and ECM) or hemidesmosomes (between epithelium and basement membrane). These clusters are involved in signal transduction. Some integrins also bind to specific adhesion molecules on other cells.
110
How does the composition of ECM alter integrin activity?
The composition of the ECM will determine which integrin complexes bind and which signals it receives – this can alter the phenotype of the cell.
111
What happens to integrin confirmation which determines activity?
* Integrins can adopt flexed and extended leg compositions.
* Switching between these confirmations affects their ability to bind their ligands, and their signalling.
* In this way, cell-ECM adhesion, and signals, can be switched on (extended) and off (flexed).
112
How do integrins sense the mechanical properties of their surroundings?
The amount of force that is generated at a focal adhesion depends on the FORCE GENERATED by the cytoskeleton AND the stiffness of the ECM. The force generated can open up the protein complex at the intracellular integrin domain, exposing new sites for the recruitment of different proteins, leading to different signalling.
113
How are integrins affected by intracellular signals? Called?
A signal generated inside the cell can act on the integrin complex to alter its affinity for ECM binding e.g. turn complex from flexed, inactive form, to an extended, active form. This is called INSIDE-OUT INTEGRIN SIGNALLING. e.g. in inflammation switching on adhesion of circulating leukocytes.
114
What happens to the legs of integrins upon activation and binding with ECM?
Remember, the legs are the intracellular domains of integrin complexes. When the integrin complex becomes activated, the legs OPEN – this exposes binding sites for the recruitment of cytoplasmic signalling proteins. When ECM ligand binds, it causes FURTHER OPENING of legs and exposes even more binding sites.
115
How does cell density affect proliferation?
Cells compete for growth factors in order to proliferate. Cells tend to form MONOLAYERS – once a monolayer has been formed, they stop proliferating. Too many cells for too little growth factor means that proliferation STOPS – called DENSITY-DEPENDENCE OF CELL DIVISION.
116
How does effect of ECM and cell density interact to determine cell proliferation? !!!
• There is crosstalk between ECM and growth factor signalling. • BOTH trigger cell proliferation by ALTERING GENE EXPRESSION through identical signalling pathways such as the MAPK pathway. • Individually, this activation is weak and transient. • When both stimuli are present, activation is strong and sustained enough to stimulate cell proliferation i.e. BOTH signals are need for efficient stimulation of proliferation – they act SYNERGISTICALLY.
117
What are the two different types of interactions between cells? How do cell-cell responses differ? (x2 actions for each)
SHORT-TERM INTERACTION: these describe circumstances such as a cell moving past another cell – cell-cell junctions form but are NOT STABLE, and they ‘repel’ one another by paralysing motility at the contact site, promoting the formation of a motile front at another site of the cell, and moving off in the opposite direction; LONG-TERM INTERACTION: these describe interactions e.g. epithelial lining formation – these stable interactions result in formation of cell-cell junctions; in addition, there is contact-induced spreading of epithelial cells where contact leads to spreading of both cells (see photo).
118
What response called where there is a change in cell motility from transient cell-cell interaction? Purpose?
Contact inhibition of locomotion. It prevents multilayering of cells!
119
What cell-cell junctions are formed in long-term cell-cell contacts? (x2 (x1 and x3)) In which cells does this take place? (x3)
Junctions are usually arranged as continuous belts (zonula/adherens junctions – cell junctions that are linked to the actin cytoskeleton and usually appear as bands encircling the cell, such as cadherins) or discrete spots (macula – such as gap junctions, desmosomes and tight junctions). Occurs in epithelial and endothelial cells which form layers, and neurones forming synapses.
120
What is the importance of contact-induced spreading of epithelial cells?
Creates a stable monolayer.
121
What is the effect of cell-cell adhesions on cell proliferation? Called? What else is required? !
Cell-cell junctions result in LOW PROLIFERATION – this is because of CONTACT INHIBITION OF PROLIFERATION. However, calcium is also required to keep proliferation low. This is because cell-cell junctions are calcium-dependent. When calcium is present, cell-cell junctions form, MAPK pathway is inactivated, and there is increased p27-KIP1. Result = LOW PROLIFERATION.
122
How is cadherin organised?
Adheren junctions are cell junctions that are LINKED TO THE ACTIN CYTOSKELETON and usually appear as bands encircling the cell. Cadherins are an example of an adheren junction: They are calcium dependent, homophilic cell adhesion molecules and bind to cadherins on other cells. They are linked to beta and alpha catenin intracellularly, which allows for recruitment of actin.
123
What are the mechanisms of contact inhibition proliferation? (x3)
• BETA-CATENIN is bound to cadherin junctions (and degraded in cytoplasm by APC complex). In the cytoplasm, when beta-catenin is not degraded, beta-catenin binds to LEF-1 in the nucleus which results in cell proliferation. Therefore, when cadherin junctions are formed, beta-catenin is taken out of the cytoplasm and cannot carry out its proliferative effects. • Clustering of cadherins after cell-cell contact activates small GTPases which can influence proliferation. • Some growth factor receptors are associated with cell-cell junctions. This reduces their capacity to promote proliferation.
124
How does cancer link to beta-catenin? Mechanism of the disease?
* In the case of APC (Adenomatous polyposis coli – where hundreds of polyps form on the colon surface), the beta-catenin (involved in the cadherin junctions) is not degraded. This leads to cell proliferation – hence there is formation of polyps which predispose cancer.
* In adenomatous polyposis coli patients, the APC complex is inactive, so beta-catenin cannot be degraded and binds to LEF-1 in the nucleus --\> cell proliferation.
125
What is the consequence of loss of contact inhibition in cancer cells? (x4) !!!
Proliferate uncontrollably (lose density dependence of proliferation), are less adherent and will multilayer (lose contact inhibition of locomotion and anchorage dependence), and epithelial breakdown cell-cell contacts. In addition, loss of contact inhibition gives cancers the ability to metastasise.
126
What are oncogenes?
Mutant gene which promotes uncontrolled cell proliferation.
127
What are proto-oncogenes?
Normal cellular gene corresponding to the oncogene.
