Cancer Flashcards
Define metaplasia.
• A reversible change in which one adult cell type (usually epithelial)is replaced by another
adult cell type
• Adaptive
Physiological examples:
- Cervix - puberty, cervix expansion, columnar epithelium of inter-cervical canal exposed to acidic pH of vagina - columnar —> squamous
- Acid reflux from oesophagus, squamous —> columnar (Barrett’s oesophagus)
Define 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 - Not invasive
• loss of architectural orientation - not maturing in normal way
• loss in uniformity of individual cells
• nuclei: hyperchromatic, enlarged - dark nuclei because conc. of DNA increases. High nuclear: cytoplasmic ratio
• mitotic figures: abundant, abnormal, in places where not usually foun
Common in: • CERVIX - HPV infection • BRONCHUS - Smoking • COLON - UC (ultra colitis) • LARYNX - Smoking • STOMACH -Pernicious • OESOPHAGUS- Acid reflux
Low grade vs high grade
Low grade:
1) risk of progression is low
2) more likely to be reversible
High grade:
1) risk of progression is high
2) less likely to be reversible
3) darker because nuclei greater
Define neoplasia, tumour, malignancy.
An abnormal, autonomous proliferation of cells unresponsive to normal growth control mechanisms
Describe the differences between benign and malignant tumours.
Benign:
1) do not invade; do not metastasise
2) encapsulated - sharp edge, fibrous capsule - easier to remove
3) usually well differentiated - look like where they came from
4) slowly growing
5) normal mitoses
Not fatal unless:
- In dangerous place: meninges (block between lateral and third ventricle which increases intra-cranial pressure)
- Secretes something dangerous: insulinoma
- Gets infected: bladder
- Bleeds: stomach
- Ruptures: liver adenoma
- Torts(twisted): ovarian cyst
Malignant:
1) invade surrounding tissue
2) spread to distant sites - block vessels, lymphatics
3) no capsule
4) well to poorly differentiated
5) rapidly growing
6) abnormal mitoses
Define metastasis.
A discontinuous growing colony of tumour cells, at some distance from the primary cancer.
These depend on the lymphatic and vascular drainage of the primary site
Lymph node involvement has a worst prognosis
E.g. Dukes A- 90% (only on bowel)
Dukes C - 30% (in lymph nodes)
Describe the nomenclature of tumours.
-oma = benign mass
Benign epithelial tumours:
- of surface epithelium = papilloma e.g. skin, bladder e.g. wart from HIV
- of glandular epithelium = adenoma e.g. stomach, thyroid, colon, kidney, pituitary, pancreas
Carcinoma:
- a malignant tumour derived from epithelium e.g. through basement membrane invasion
- squamous cell
- adenocarcinoma
- transitional cell e.g. bladder
- basal cell carcinoma e.g. skin
Benign soft tissue tumours:
E.g. osteomalacia
Leiomyoma - smooth muscle
Sarcoma:
- a malignant tumour derives from connective tissue (mesenchymal) cells
- fat = liposarcoma
- bone = oestosarcoma
- cartilage = chondrosarcoma
- muscle, striated = rhabdomyosarcoma, smooth = leiomyosarcoma
- nerve sheath = malignant peripheral nerve sheath tumour
Leukaemia and lymphoma:
-tumours of white blood cells:
Leukaemia = a malignant tumour of bone marrow derived cells which circulated in the blood
Lymphoma = a malignant tumour of lymphocytes (usually) in lymph nodes (End in -Oma but malignant)
Teratoma:
-tumour derives from germ cells, which have the potential to develop into tumours of all three germ cell layers:
1) ectoderm
2) mesoderm
3) endoderm
Can develop into any type of tissue
-gonadal teratomas in males, all malignant
-gonadal teratomas in females, most are benign
Hamartoma
-localised overgrowth of cells and tissues native to the organ
-cells are mature but architecturally abnormal
-common in children, and should stop growing when they do
E.g. bile duct hamartomas, bronchial hamartomas
Describe the differentiation of tumours.
Criteria for assessing differentiation of a malignant tumour:
-evidence of normal function still present and production of:
Keratin, mucin, bile, hormones - does it still produce these?
Various garden systems for cancer of breast, prostate, colon e.g. Gleason grading system for prostate
No differentiation = anaplastic carcinoma
Describe the TNM system.
The grade of a tumour describes its degree of differentiation.
The stage of a tumour describes how far it has spread.
Tumours of higher grade (I.e. poorly differentiated) tend to be of higher stage (I.e. spread further)
Overall stage is more important than grade in determining prognosis.
T=tumour
N-node
M=metastasis
State the factors which influence the rate of cell division.
1) embryonic vs adult cells - embryo faster
2) complexity of system e.g. yeast faster
3) necessity for renewal (intestinal epithelial cells faster than hepatocytes - queiescent, if only injury is it highly proliferative)
4) state of differentiation (some cells never divide e.g. neurones and cardiac myocytes
5) tumour cells
Describe the relevance of the appropriate regulation of cell division.
Premature, aberrant mitosis results in cell death.
In addition to mutations in oncogenes and tumour suppressor genes, most solid tumours are aneuploid (abnormal chromosome number and content)
Various cancer cell lines show chromosome instability (lose and gain whole chromosomes during cell division)
Perturbation (deviation from normal state) of protein levels of cell cycle regulators is found in different tumours - abnormal mitosis
Contact inhibition of growth
Attacking the machinery that regulates chromosome segregation is one of the most successful anti-cancer strategies in clinical use.
Explain the cell cycle.
Orderly sequence of events in which a cell duplicates its contents and divides into two.
- duplication
- division
- co-ordination
Interphase (duplication)
- DNA
- organelles and protein synthesis
M-phase: mitosis (division)
- nuclear division
- cell division (cytokinesis)
Mitosis - most vulnerable period of cell cycle:
- cells are more easily killed (irradiation, heat shock, chemicals)
- DNA damage can not be repaired
- gene transcription silences
- metabolism reduced, cell’s energy focussed on division
Interphase:
G0 = cell cycle machinery dismantled
G1 phase (Gap) = decision point, like checkpoint - are all organelles duplicated?
S phase - synthesis of DNA/ protein
- DNA replication
- protein synthesis: initiation of translation and elongation increased; capacity is also increased (increase in ribosomes)
- replication of organelles (centrosomes, mitochondria, Golgi etc.) in the case of mitochondria, needs to coordinate with replication of mitochondrial DNA
G2 phase (Gap) - decision point - check DNA duplication, mutation
Mitosis:
Prophase
-condensation of chromatin
-Condensed chromosomes each consists of 2 sister chromatids, each with a kineticochore and joined by centromere
-duplicated centrosomes migrate to opposite sides of the nucleus and organise the assembly of spindle microtubules
-mitotic spindle forms outside nucleus between the 2 centrosomes
Spindle formation - radial microtubule arrayed (ASTERS) form around each centrosome (microtubule organising centres - MTOC), radial arrays meet, polar micro tubes form - stabilised at centre of cell
Microbes are in a dynamic state
Early prometaphase
- breakdown of nuclear membrane
- spindle formation largely complete
- attachment of chromosomes to spindles via kinetochores (centromere region of chromosomes) - microtubules capture at this region
Late prometaphase:
- microtubule from opposite pole is captured by sister kinetochore
- chromosomes attached to each pole congress to the middle
- chromosome slides rapidly towards center along microtubules
Metaphase
Chromosomes aligned at equator of the spindle
Anaphase
Paired chromatids separate to form two daughter chromosomes
Cohesion holds sister chromatids together
Anaphase A and B
Anaphase A:
Breakdown cohesion
Micro tubes get shorter
Daughter chromosomes pulled toward opposite spindle poles
Anaphase B:
1-daughter chromosomes migrate towards poles
2-spindle poles ‘centrosomes’ migrate apart
Telophase
- daughter chromosomes arrive at spindle
- nuclear envelope reassembles at each pole
- assembly of contractile ring - cleavage furrow
Cytokinesis
- new membrane inserted
- acto-myosin ring contracts
- midbody begins to form
- interphase microtubule array reassembles
- chromatin decondenses and nuclear sub structures reform
Transition out of metaphase: spindle assembly checkpoint
-senses completion of chromosome alignment and spindle assembly (monitors kinetochore activity) - make sure in correct position e.g. equator and all attached to microtubules
Requires:
CENP-E - tension
BUB protein kinases - BUBs dissociate from kinetochore when chromosomes are properly attached to the spindle and when all dissociated, anaphase proceeds.
Describe how aneuploidy occurs.
Mis-attachment of microtubules to kinetochores
1) synthelic attachment: both sister chromatids attached but attached to wrong microtubules - same daughter cell in this case ; both sister chromatids at same pole
2) monotelic attachment - only one of the sister chromatids attached to the kinetochore
3) merotelic attachment - more than one microtubule to same sister chromatid; chromosome loss at cytokinesis
4) amphelic attachment - normal
Aberrant centrosome/ DNA duplication
1) aberrant cell cycle - DNA and centrosome duplication —> 4 centrosomes
2) aberrant cytokinesis from multipolar spindle, chromosomes don’t know where to go
Describe anti-cancer therapy by inducing gross chromosome mis-segregations.
Checkpoint kinase (CHKE1 and CHKE2) - serine threonine kinase activation holds cells in G2 phase until all is ready inhibition leads to ultimately cell transition to mitosis
Taxanes and vinca alkaloids (breast and ovarian cancers)
- alters microtubule dynamics
- produces unattached kinetochores
- causes long-term mitotic arrest
What happens if something goes wrong during the cell cycle?
1) Cell cycle arrest
- at check points (G1 and spindle check point)
- can be temporary (I.e. following DNA repair)
2) Programmed cell death (apoptosis)
-DNA damage too great and cannot be repaired
-chromosomal abnormalities
-toxic agents
Cell cycle progression aborted and cell destroyed
Describe the effects fo tumours on checkpoints.
1) G1 checkpoint - cells grow
2) G2 checkpoint - DNA damage not checked
3) Metaphase checkpoint - don’t check sister chromatids alignment
4) G0 - cell cycle apparatus not dismantled
What triggers a cell to enter the cell cycle and divide?
In the absence of stimulus, cells go into G0 (quiescent phase)
Exit from G0 highly regulated - requires growth factors and intracellular signalling cascades.
Signalling cascades:
- response to extracellular factors
- signal amplification
- signal integration
- modulation by other pathways
- regulation of divergent responses
How does protein phosphorylation after protein function?
Causing a change in shape (conformation) leading to change in activity (+ve or -ve)
Creating a docking site for another protein
In presence of ligand:
- receptors form diners
- are activated by phosphorylation
Receptor activation triggers:
-kinase cascades
-binding of adapter proteins
Kinases phosphorylation, phosphatases dephosphorylate
What are the main anti-cancer treatment modalities?
Surgery
Radiotherapy
Chemotherapy
Immunotherapy
What are the types of genetic mutations causing cancer?
Chromosome translocation
Gene amplification (copy number variation)
Point mutations within promoter or enhancer regions of genes
Deletions or insertions
Epigenetic alterations to gene expression
Can be inherited
Cancer is a disease of the genome
State what the systemic therapy in treating cancer involves.
Cytotoxic chemotherapy
1) alkylating agents
2) antimetabolites
3) anthracyclines
4) vinca alkaloids and taxanes
5) topoisomerase inhibitors
Targeted therapies
1) small molecule inhibitors
2) monoclonal antibodies
Explain cytotoxic chemotherapy.
Cytotoxics select rapidly dividing cells by targeting their structures (mostly the DNA)
-Given intravenously or by mouth (occasionally)
-works systemically
-non targeted - affects all rapidly diving clels in body e.g. gut mucosa, bone marrow cells —> mucositis, BM suppression
-given post-operatively: adjuvant - mop up floating residual cells
Pre-operatively: neoadjuvant - tumours that are chemosensitive are given these to downstage, can reduce huge surgery like local incision
As monotherapy it in combination with curative or palliative intent
1) Alkylating agents
-Add alkyl (CnH2n+1) groups to guanine residues in DNA
-Cross-link (intra, inter, DNA-protein) DNA strands and prevents DNA from uncoiling at replication
-Trigger apoptosis (via checkpoint pathway)
-Encourage miss-painting - oncogenes (secondary malignancy)
E.g. chlorambucil, cyclophosphamide, dacarbazine, temozolomide
2) Pseudo-alkylating agents
-Add platinum to guanine residues in DNA
-Same mechanism of cell death as alkylating agents
E.g. carboplatin, cisplatin, oxaliplatin
Side effects of 1) and 2)cause hair loss (not carboplatin), nephrotoxicity, neurotoxicity e.g. peripheral neuropathy, ototoxicity (platinums), nausea, vomiting, diarrhoea, immunosuppression, tiredness
3) Anti-metabolites
-Masquerade as purine or pyramiding residues leading to inhibition of DNA synthesis, DNA double strand breaks and apoptosis.
-Block DNA replication (DNA-DNA) and transcription (DNA-RNA)
-Can be purine (adenine and guanine), pyramiding (thymine/ uracil and cytosine) or folate antagonists (which inhibit dihydrofolate reductase required to make folic acid, an important building block for all nuclei acids - especially thymine)
E.g. methotrexate (folate), 6-mercaptopurine, decarbazine and fludarabine (purine), 5-fluorouracil, capecitabine, gemcitabine (pyramidine)
Side effects
Hair loss (alopecia) – not 5FU or capecitabine Bone marrow suppression causing anaemia, neutropenia and thrombocytopenia
Increased risk of neutropenic sepsis (and death) or bleeding
Nausea and vomiting (dehydration)
Mucositis and diarrhoea
Palmar-plantar erythrodysesthesia (PPE) - red hands and red feet and skin begins to peel
Fatigue
4) anthracyclines
-inhibit transcription and replication by intercalating (I.e. inserting between) nucleotides within the DNA/ RNA strand
-also block DNA repair -mutagenic
-they create DNA and cell membrane damaging free oxygen radicals
E.g. doxorubicin, epirubicin
Side effects: • Cardiac toxicity (arrythmias, heart failure) – probably due to damage induced by free radicals • Alopecia • Neutropenia • Nausea and Vomiting • Fatigue • Skin changes • Red urine (doxorubicin “the red devil”)
5) Vinca alkaloids and taxanes
- originally derived from natural sources
- work by inhibiting assembly (vinca alkaloids) or disassembly (taxanes) of mitotic microtubules causing dividing cells to undergo mitotic arrest
Side effects:
(Of microtubule targeting drugs in general too)
•Nerve damage: peripheral neuropathy, autonomic neuropathy
•Hair loss
•Nausea
•Vomiting
•Bone marrow suppression (neutropenia, anaemia etc)
•Arthralgia
•Allergy
6) Topoisomerase inhibitors
-Topoisomerases are required to prevent DNA torsional strain during DNA replication and transcription
-They induce temporary single strand (topo1) or double strand (topo2) breaks in the phosphodiester backbone of DNA
-they protect the free ends of DNA from aberrant recombination events
Anthracyclines cause permanent DNA damage
E.g. Topotecan and irinotecan (topo I) and etoposide (topo II) alter binding of the complex to DNA
Side effects: • (irinotecan): Acute cholinergic type syndrome – diarrhoea, abdominal cramps and diaphoresis (sweating). Therefore given with atropine • Hair loss • Nausea, vomiting • Fatigue • Bone marrow suppression
If treatment will reduce someone’s chance of relapse with the disease by 30% and chance of dying by 20%. Is it worth the toxicity?
Yes because side effects can be controlled by drugs and not all are frequent.
State some resistance mechanisms against chemotherapy.
DNA repair mechanisms upregulated and DNA damage is repaired —> stopping DNA double strand breaks
DNA adducts replaced by Base Excision repair (using PARP)
Drug effluxed from the cell by ATP-binding cassette (ABC) transporters.
Describe the problem present in non-monogenic cancers.
You can cut the wiring in monogenic cancers but for others parallel pathways or feedback cascades are activated
Nowadays we have dual kinase inhibitors which can prevent feedback loops but increase toxicities.
What are the hallmarks of cancer?
Used to be 6 now 10. First 6 is original
- Self –sufficient
• Normal cells need growth signals to move from a
quiescent (resting) to active proliferating state • These signals are transmitted into the cell via growth
factors binding transmembrane receptors and
activating downstream signalling pathways - Insensitive to anti-growth signals
- Anti-apoptotic
- Pro-invasive and metastatic
- Pro-angiogenic
- Non-senescent
- Dysregulated metabolism
- Evades the immune system
- Unstable DNA
- Inflammation
What makes up a growth factor receptor?
Top to bottom: Ligand-binding site Membrane Kinase domain C-terminal region with lots of tyrosine (for autophosphorylation)
Explain the over-expression of receptors in cancers.
HER2 – amplified and over-expressed in 25% breast cancer
EGFR – over-expressed in breast and colorectal cancer, lung cancer
PDGFR- glioma (brain cancer)
VEGF – prostate cancer, kidney cancer, breast cancer
FGFR (head and neck cancers, myeloma)
Therefore increased kinase cascade and signal amplification
Explain targeted therapies.
1) Monoclonal antibodies
- momab (derived from mouse antibodies)
- ximab (chimeric) e.g cetuximab
- zumab (humanised) e.g. bevacizumab trastuzumab
- mumab (fully human) e.g. panitumumab
Humanized monoclonal antibody, murine regions (black) interspersed within the light (light gray) and heavy (dark gray) chains of the Fab portion.
Chimeric antibody murine component (black) of the variable region of the Fab section is maintained integrally.
Monoclonal antibodies target the extracellular component of the receptor
Neutralise the ligand
Prevent receptor dimerisation
Cause internalisation of receptor
Also activate Fcgamma-receptor-dependent phagocytosis or cytolysis induces complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity
Examples: bevacizumab binds and neutralised VEGF, improving survival in colorectal cancer
Cetuximab targets EGFR
2)Small molecule inhibitors
Bind to the kinase domain of the tyrosine kinase within the cytoplasm and block autophosphorylation and downstream signalling.
Small molecule inhibitors act on receptor TKs but also intracellular kinases - therefore can affect cell signalling pathways
Examples of SMIs inhibiting receptors:
erlotinib (EGFR), gefitinib (EGFR), lapatinib (EGFR/HER2), sorafinib (VEGFR)
SMIs inhibiting intracellular kinases:
Sorafinib (Raf kinase) Dasatinib (Src kinase) Torcinibs (mTOR inhibitors)
By acting on receptors (either externally or internally), targeted therapies block cancer hallmarks (e.g VEGF inhibitors alter blood flow to a tumour, AKT inhibitors block apoptosis resistance mechanisms) WITHOUT the toxicity observed with cytotoxics
BUT CAN DEVELOP RESISTANCE AND EXPENSIVE
Describe the resistance mechanisms against targeted therapies.
• Mutations in ATP-binding domain (e.g BCR-Abl fusion gene (Philadelphia) and ALK gene, targeted by Glivec - imatinib and crizotinib respectively)
• Intrinsic resistance (herceptin effective in 85% HER2+ breast cancers, suggesting other driving
pathways)
• Intragenic mutations
• Upregulation of downstream or parallel pathways
Describe anti-sense oligonucleotides and RNA interference.
• Single stranded, chemically modified DNA-like molecule 17-22 nucleotides in length
• Complementary nucleic acid hybridisation to target gene hindering translation of specific
mRNA
• Recruits RNase H to cleave target mRNA
• Good for “undruggable” targets
RNA interference
• Single stranded complementary RNA
• Has lagged behind anti-sense technology –
especially in cancer therapy
• Compounds have to be packaged to prevent
degradation - nanotherapeutics
• CALAA-01 targeted to M2 subunit of
ribonucleotide reductase. Phase I clinical trials in cancer –results awaited
What is the restriction point?
It is found in G1 and cell monitors its own size and external signals.
The cell is now committed in the cell cycle and does not require any more GF’s.
What is c-Myc?
An oncogene over expressed in many tumours
A transcription factor which stimulates the expression of cell cycle genes and is need to enter S phase
Describe adaptor proteins.
Tyrosine phosphorylation provides docking sites for adapter proteins
Protein-protein interactions: protein binding – bringing proteins together
Proteins are modular and
contain domains, i.e. functional and structural units that are copied in many proteins
Some domains are important in
molecular recognition – have no enzymatic function of their own, simply bring other proteins together
E.g. Grb2 binds to SH3 - proline-rich regions (constitutive) and SH2 (phosphorylation tyrosine (transient)
What activates and inactivated RAS?
How can RAS be oncogenically activated by mutations?
1) exchange factors e.g. SOS activates
2) GTPase activating proteins inactivate
RAS must be bound to plasma membrane to be activated
Oncogenically activated:
To increase the amount of active GTP-loaded Ras by:
-preventing GAP binding
-preventing GTP hydrolysis
What is ERK?
Extracellular signal-regulated kinase
It’s a type of MAPK cascade (mitogen-activated protein kinase)
Describe Cdks and cyclins.
Cdks (cyclin-dependent kinases)
Present in proliferating cells throughout cell cycle
Activity is regulated by:
-interaction with cyclins
-phosphorylation
Cyclins
- transiently expressed at specific at specific points in the cell cycle
- regulated at level of expression
- synthesised then degraded
Cyclins bind to and activate Cdk(s) triggering different events in the cell cycle
Cdks phosphorylation proteins (on serine or threonine) to drive cell cycle progression
E.g. nuclear lamins (causes breakdown of nuclear envelope)
E.g. retinoblastoma protein (pRb) - tumour suppressor - inactivated in many cancers
Regulation of Cdks by phosphorylation
Requires activating phosphorylation AND removal of inactivating phosphorylation
Cyclins activate Cdks but also alter substrate specificity
Substrate accessibility changes through cell cycle
Cyclical activation - Cdks become sequentially active and stimulate synthesis of genes required for next phase
Describe what happens at the anaphase checkpoint in the metaphase-anaphase transition.
Cdk1/cycB active. Mitosis on hold - key substrates phosphorylated
Signal from fully attached kinetochores causes cyclin B to be degraded:
- Cdk1 inactivated
- key substrates dephosphorylated
- mitosis progresses
Describe the two CKI families.
INK4 family and CIP/KIP family
G1 phase CKIs - inhibit Cdk4/6 by displacing cyclin D
S phase CKIs - inhibit all Cdks by binding to the Cdk/cyclin complex
CKI must be degraded to allow cell cycle progression
Describe cell cycle regulatory proteins and cancer.
Oncogenes (derived from normal proto-oncogenes)
-EGFR/HER2, mutationally activated or overexpressed in many breast cancers (herceptin antibody for the treatment of HER2-positive metastatic breast cancer)
-Ras, mutationally activated in many cancers (inhibitors of membrane attachment)
-cyclin D1, over expressed in 50% of breast cancers
-B-Raf, mutationally activated in melanomas (kinase inhibitors in trials)
-c-Myc, overexpressed in many tumours
Higher up in signal, harder to treat because affects more pathways
Tumour suppressors
- RBC, inactivated in many cancers
- P27KIP1, underexpression correlates with poor prognosis in many malignancies
What are the types of DNA damage?
Carbon ring structures can be easily activated chemically because chemically reactive
Also can interchange easily e.g. thymine —> uracil by removal of methyl group.
Base modifications prevent replication or cause mutations:
Deamination - the primary amino groups of nucleic acid bases are somewhat unstable. They can be converted to ketogroups in reactions like cytosine to uracil
Chemical modification - e.g. hyper-reactive oxygen (hydrogen peroxide, peroxide radicals .etc.) can modify DNA bases. A common product of thymine oxidation is thymine glycol.
Hyper-reactive oxygen species are also generated by ionising radiation (x-rays, gamma rays)
Many environmental chemicals can modify DNA bases (including food), frequently by addition of a methyl or other alkyl group (alkylation). Addition of larger molecules defines “adducts” - carcinogens can cause them
Photodamage - ultraviolet light is absorbed by the nuclei acid bases, and the resulting influx of energy can induce chemical changes. The most frequent photo products are the consequences of bond formation between adjacent pyramid Ines with one strands (intra-DNA damage) e.g. thymidine react with each other to form thiamine dimer
Nick - radiation can break phosphodiester bond
Gap - lots of breaks/nicks
Thymine dimer - distortion of helix
Base pair mismatch - because of change in base
What are the causes of DNA damage?
Chemicals (carcinogens)
- dietary
- lifestyle
- environmental
- occupational
- medical
- endogenous - metabolisms??
Radiation
-ionising
Generates free radicals in cells including oxygen free radicals - super oxide radical O2• , hydroxyl radical HO•
Possess unpaired electrons - electrophilic and therefore seek out electron rich DNA - cause nicks e.g. ring-opened guanine and adenine
-solar
Form pyramidine (thymine) diners - skin cancer
-cosmic
Damage from carcinogens:
- DNA adducts and alkylation
- Base diners and chemical cross-links
- Base hydroxylations (reactive oxygen) and abasic sites formed (base destroyed but DNA structure underlying it intact)
The importance of DNA damage:
- DNA damage can lead to mutation
- mutation may lead to cancer
- damaging DNA is an important strategy in cancer therapy (chemotherapy)
Explain the role of metabolism in DNA damage.
Mammalian metabolism
Phase I - liver
-addition of functional groups e.g. oxidation, reduction, hydrolysis
-mainly cytochrome p450 mediated
Phase II
-conjugation of phase I functional groups e.g. sulphation, glucoronidation, acetylation, methylation, amino acid and glutathione conjugation (generates polar -water soluble metabolites)
Polycyclic aromatic hydrocarbons:
-common environmental pollutants
-formed from combustion of fossil fuels and tobacco
E.g.
Two step epoxidation of B[a]P - mutation in lung cancer causes by tobacco smoke
Epoxidaiton of aflatoxin B1 - formed by Aspergillus flavus mould, stored peanuts
Metabolism of 2-napthylamine - past components of dye-stuffs, bladder cancer - nitrenium ion end up in urine and change pH causing cancer
Explain the repair of DNA damage.
The greater the persistence of damage then the greater the chance of mutagenic event.
P53 is a tumour suppressor gene, p53 is kept inactive by MDM2. MDM2 lost if p53 activated
P53 is a TF so can turn on different pathways to respond to damage e.g. DNA repair in response to DNA replication stress, double-strand breaks .etc.
Types of DNA repair:
- direct reversal of DNA damage
- Base excision repair (mainly for apurinic/apyrimidinic damage - lost base)
- nucleotide excision repair (mainly for bulky DNA adducts)
- during or post replication repair
- DNA mismatch repair
DNA mismatch repair:
-mismatches that arise during replication are corrected by comparing the old and new strands (proof-reading)
Bulge in DNA = wrong base, recognised by MSH and MCH protein, bind to bulge, nucleus cuts out base and then polymerase restores correct sequence
-other systems deal with mismatches generated by base conversions such as those which result from deamination
Direct DNA repair:
Involves the reversal or simple removal of the damage by the use of proteins which carry out specific enzymatic reactions
E.g. photolyases (activated by normal light, not UV) repair thymine diners
Excision repair of DNA damage:
For nicks
1) Damage to G but not to phosphodiester backbone
2) remove base without affecting phosphodiester bone by DNA-glycosylase
3) AP-endonuclease cuts open nuclear strand
4) DNA polymerase adds correct base
5) DNA lipase
For gaps, e.g. caused by big adduct groups
1) damage to both base and DNA
2) endonuclease - forms nicks around mutation
3) helicase removes phosphodiester bond and bases
4) DNA polymerase corrects base
5) DNA ligase
Double strand break repair:
Double-strand breaks are made:
• Under physiological conditions during
somatic recombination and transposition. e.g.
Transient base pairing of V(D)J recombination
• During Homologous Recombination.
• As a result of ionizing radiation and oxidative
stress induced DNA damage.
1) following a double sytrand break, a 3’-5’ exonuclease exposes a 5’ single-strand overhand until it finds a sequence which matches together
2) transient base pairing of several nucleotides enables ends to come together
3) DNA polymerisation
4) nucleolytic processing
5) ligation
Non-homologous repair- if oevrlangs font match, Ku proteins hold DNA together forcing repair
Summary:
Direct reversal of DNA damage
▪ photolyase splits cyclobutane pyrimidine-dimers
▪ methyltransferases & alkyltransferases remove alkyl groups from bases
Base excision repair (mainly for apurinic/apyrimidinic damage)
▪ DNA glycosylases & apurinic/apyrimidinic endonucleases + other
enzyme partners
▪ A repair polymerase (e.g. DNA Polb) fills the gap and DNA ligase
completes the repair.
Nucleotide excision repair (mainly for bulky DNA adducts)
▪ Xeroderma pigmentosum proteins (XP proteins) assemble at the damage
.A stretch of nucleotides either side of the damage are excised.
▪ Repair polymerases (e.g. DNA Pold/b) fill the gap and DNA ligase complete the repair.
During- or post-replication repair
▪ mismatch repair
▪ recombinational repair
What are some human genetic disease involving nucleotide excision repair (NER)?
- Xeroderma Pigmentosum
- severe light sensitivity
- severe pigmentation irregularities
- early onset of skin cancer at high incidence • elevated frequency of other forms of cancer • frequent neurological defects
Trichothiodystrophy • sulphur deficient brittle hair • facial abnormalities • short stature • ichthyosis (fish-like scales on the skin) • light sensitivity in some cases Cockayne's syndrome • dwarfism • light sensitivity in some cases • facial and limb abnormalities • neurological abnormalities • early death due to neurodegeneration
What are the consequences of DNA damage?
Normally:
Carcinogen damage leading to altered DNA —> apoptosis —> cell death
Carcinogen damage leading to altered DNA —> efficient repair —> normal cell
Problem:
Carcinogen damage leading to altered DNA —> incorrect repair/ altered primary sequence —> DNA replication and cell division: fixed mutations —> transcription/translation giving aberrant proteins OR carcinogenesis if critical targets are mutated: oncogenes, tumour suppressor genes
Give examples of therapeutic agents which cause DNA damage.
Alkylating agents - alkylate to cause DNA damage and apoptosis
Agents that make bulky agents e.g. cisplatin
Agents that induce double strand breaks e.g. ionising radiation
Explain carcinogen testing.
Testing for DNA damage:
in vitro bacterial gene mutation assay —> in vitro mammalian cell assay —> in vivo mammalian assay —> investigate in vivo mammalian assay
Bacterial (Ames) test for mutagenicity of chemicals
1) chemical to be tested and rat liver enzyme preparation-s9 (cytochrome p450 activity) added into bacteria that do not synthesis histidine(aa required to grow) e.g. salmonella strain -mutation
2) conversion of chemical to active metabolism
3) carcinogenic agents may correct defective mutation in salmonella, bacteria acquire ability to synthesise histidine and colonies can be seen on Petri dish
Detecting DNA damage in mammalian cells -chromosomal aberrations:
Treat mammalian cells with chemical in presence of liver s9. Look for chromosomal damage
In vitro micronucleus assay:
Cells treated with chemical and allowed to divide
Binucleate cells assessed for presence of micronuclei
Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)
If DNA becomes very damaged, it gets budded as micronucleus
Murine bone marrow micronucleus assay:
Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei
Why does programmed cell death occur?
1) harmful cells (e.g. cells with viral infection, DNA damage)
2) developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens)
3) excess/ unnecessary cells: (embryonic development: brain to eliminate excess neurones; liver regeneration; sculpting of digits and organs)
4) obsolete cells (e.g. mammary epithelium at the end of lactation)
5) exploitation - chemotherapeutic killing of cells
Describe the difference between necrosis and apoptosis.
Necrosis - unregulated cell death associated with trauma, cellular disruption and an inflammatory response
Apoptosis (programmed cell death) - regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response
Necrosis:
- plasma membrane becomes permeable
- cell swelling and rupture of cellular membranes, organelles swell, chromatin condenses
- release of proteases leading to autodigestion and dissolution of the cell
- localised inflammation
Apoptosis:
Latent phase - death pathways are activated, but cells appear morphologically the same
Execution phase
- loss of microvilli and intercellular junctions - permeability of epithelium compromised
- cell shrinkage, epithelium closes around - permeability resorted
- loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer leaflet) - imbalance in lipid composition
- chromatin and nuclear condensation
- DNA fragmentation
- Formation of membrane blebs
- fragmentation into membrane-enclosed apoptotic bodies
- apoptosis bodies phagocytosis by neighbouring cells and macrophages
Plasma membrane remains intact - no inflammation
Apoptosis-like PCD - some, but not all, features of apoptosis. Display of phagocytise recognition molecules before plasma membrane lysis
Necrosis-like PCD - variable features of apoptosis before cell lysis; “aborted apoptosis”
What are the mechanisms of apoptotic cell death?
- The executioners – Caspases
- Initiating the death programme
• Death receptors
• Mitochondria - The Bcl-2 family
- Stopping the death programme
1) cysteine-dependent aspartate-directed proteases - cleave at specific sites
-executioners of apoptosis
-activated by proteolysis
-cascade of activation
Inactive at start - regulation, autofolded on itself
Initiator and effector; initiator = trigger apoptosis by cleaving and activating; effector = carry out the apoptotic programme
Cleave at:
- CARD (cascade recruitment domain)
- DED (death effector domain)
- homotypic (same type) protein-protein interactions e.g. cascade would dimerise with cascade
Describe how effector caspases execute the apoptotic programme.
Cleave and inactivate proteins or complexes (e.g. nuclear lamins leading to nuclear breakdown) -loss of function
Activate enzymes (incl. protein kinases; nucleases .e.g. Caspase-Activated DNase, CAD) by direct cleavage, or cleavage of inhibitory molecules - gain of function
Both the above apply to monomeric substrates and multiprotein complexes
What are the two mechanisms of caspase activation?
Death by design - receptor-mediated (extrinsic) pathways
Death by default - mitochondrial (intrinsic) death pathway
State the Bcl-2 family proteins.
Anti-apoptotic - promote cell survival (mitochondrial)
Bcl-2
Bcl-xL
Pro-apoptotic (move between cytosol and mitochondria) Bid Bad Bax Bak
How does PKB/Akt induce cell survival by blocking apoptosis?
- Phosphorylates and inactivates Bad 2. Phosphorylates and inactivates caspase 9
- Inactivates FOXO transcription factors (FOXOs promote
expression of apoptosis-promoting genes) - Other, e.g. stimulates ribosome production and protein
synthesis
Describe inhibitor of apoptosis proteins (IAPs).
They regulate programmed cell death in the extrinsic pathway by:
- binding to procaspases and preventing activation
- binding to active caspases and inhibit their activity
State the cytoprotective/ anti-apoptotic pathways.
Bcl-2, Bcl-xL: intrinsic pathway
FLIP, IAPs: extrinsic pathway
Growth factor pathways via PI3’K and PKB/Akt
