RadBio Flashcards
Phases of cell cycle.
What is the role of each
1) G1 = Period of metabolic activity, growth and repair,
2) S= DNA synthesis
3) G2 = Confirm accurate replication before M phase
4) M (broken into subphases of Mitosis and finally exocytosis)
Phases of mitosis
Paedo Priests Meet At The Cathedral
1) Prophase:
■ condensation of chromosomes, each containing two sister chromatids
■ moving apart of centrosomes
■ assembly of mitotic spindle in between centrosomes
2) prometaphase
■ breakdown of nuclear envelope
■ chromosomes attach to spindle microtubules and undergo active movement.
3) metaphase
■ chromosomes are aligned at the equator of the spindle
4) anaphase:
■ sister chromatids separate and pulled towards spindle pole
■ the spindle poles also move apart.
5) telophase:
■ chromosomes arrive at the spindle poles
■ new nuclear envelope reassembles- forming 2 nuclei
Cytokenesis
● Mitosis ends with formation of two nuclei and beginning of cytokinesis
○ cytoplasm is divided in two by a contractile ring
○ pinched into two daughters
DNA damage response activate x distinct checkpoints?
Where and what are they called?
There are 4 (remember DEAB 4(6), 2, 2,(1) 1)
1) G1: Cyclin D-CDK4, Cyclin E-CDK2
2) S: the interphase check point - Cyclin A - CDK2 (the initiator of replication)
3) G2: Cylina - now paired with CDK1 which activates cyclin B-CDK1 - signal to start making spindles as well as progress to M phase.
Checkpoint activation requires inhibition of:
And
by what 2 broad pathways?
Checkpoint activation requires inhibition of cyclin-CDK complex:
○ by activation of CDK inhibitors
○ by affecting phosphorylation and activity of the CDK itself.
What is E2F? How is it activated?
E2F is the main G1/S checkpoint regulator
● In G1 E2F is bound to Rb protein.
● Phosphorylation of Rb by Cyclin D-CDK 4 and Cyclin E-CDK 2 releases E2F
● Released E2F stimulates Cyclin E production, initiating DNA replication and therefore S phase
For IR induced damage what is the key pathway for cycle arrested in G1?
Double stranded break:
1) Sensor = MRN complex
2) Transducer = ATM
3) Activators gammaH2Ax, BRAC1
4) CHK2 phosphorylated (see below)
5) stabilisation and activation p53: DNA repair, upregulates p21, cell arrest (via p21), apoptosis
Direct action of p21: Inhibit cyclin D-CDK4 complex
Direct action of CHK2: Inhibit CDC25CC which maintains CDK2 (i.e leads to DEphosphorylaed = inactivated CDK2)
CHK1 also does this
inhibtion and release of E2F)
The S “intra phase” checkpoint does what?
Delays DNA replication and initiates repair.
The S phase checkpoint is regulated by?
Check point is activated by?
● Regulated by ATM and ATR
● Checkpoint activated by phosphorylation and activation of CHK1 and CHK2 proteins.
What does CDC stand for?
What do they do?
Cell Division Cycle Phosphatases (CDC)
● Multiple types of CDC.
○ CDC25A, B and C
● Remove phosphate groups from cyclin-CDK complexes and activating them.
● Inactivation of CDC renders cyclin-CDK complexes inactive.
What does CDC25CC do?
CDC25CC maintains activity of (dephophorylates) CDK2
Activation of CHK 1 and 2 causes what?
● CHK1/2 phosphorylate and inactivate CDC25CC which maintains activity (dephosphorylation) of CDK2
● As a result, there is increased phosphorylated (inactive) CDK2 and cell cycle does not progress.
● BRCA1/2 also plays a role in DNA repair (HR)
How do the concentrations of cyclins and CDK vary throughout the cell cycle?
Cyclin concentration varies for each phase while [CDK] is constant
How does cyclin D work?
Binds CDK 4 and 6 in G1, these activated kinases then phosphorylate Rb releasing E2F which increases [cyclin E)
How does cyclin E work?
Binds CDK 2 in G1, Complex does 3 things:
1) completes phosphorylation of Rb protein leading to release of E2F and increased Cyclin E concentration.
2) Phosphorylates p27 and p21, causing their proteolysis (p27 and p21 are CDK complex inactivators)
3) initiates assembly of the pre-replication complex.
What induces cyclin D?
Induced by Ras/Raf/MEK/ERK pathway
How does cyclin A work
What drives its transcription?
Binds CDK2 in S phase. and CDK 1 in early G2. Transcription driven by E2F.
○ In S-phase associates with CDK 2
■ complex initiates DNA replication
■ also inhibits the action of Cyclin E/CDK 2
○ In G2 phase associated with CDK 1
■ Involved in activation of Cyclin B
What mediator removes cylin A, what other cyclin does this mediator remove?
Destroyed by APC (anaphase promoting complex) in prometaphase. Also removes cyclin B
How does Cyclin B work? What inactivates it? What destroys it
Active in late G2, Bound to CDK 1
Complex called the mitosis promoting factor:
■ involved in expression of proteins for creation of the mitotic spindle
■ necessary to progress into M-phase
■ inactivated by p53.
Destroyed by APC (anaphase promoting complex) in prometaphase. Also removes cyclin
What are cyclin dependent kinase inhibitors?
What are the main groups? Examples from each group
Group of proteins which inhibit production or function of cyclin-CDK complexes
● Two major families:
1) INK4 proteins inhibit binding of Cyclin D to CDK4 and CDK6 causing G1 arrest. P16
2) CIP/KIP proteins bind and inhibit function of Cyclin E-CDK2 and Cyclin D-CDK4
■ P21, P27, P57
How does p16 work?
Triggered by overactivation of Ras/MAPK pathways, DNA damage ect.
Cyclin dependent kinase inhibitor INK4 gene (same locus on chromosome 9p21 as ARF) coding p16 that inhibits binding of Cyclin D to CDK4 and CDK6 causing G1 arrest.
I.e CDK-INK4 complexes rather than CDK-cyclin complexes
The “INK4” = INhibitors of cdK 4
CIP/KIP proteins are capable of inhibiting all CDKs.
How does p53 work?
Guardian of the genome
Activates P21 which inhibits CDC2 and cdk2 in addition
● Regulates pathways related to DNA repair, apoptosis, angiogenesis and cell cycle arrest.
● Activators:
○ MAPK family: membrane damage, oxidative stress, osmotic shock etc
○ ATM/CHK2/DNA-PK: DNA damage
○ P14 (via inactivation of MDM2): oncogene activation
● Loss of p53 leads to dysregulation of the cell cycle
Li-Fraumeni syndrome ?
Autosomal dominant p53 gene mutation (the gene is called Tp53). Leads to multiple tumours and onset at an early age.
Cell Cycle Kinetic Parameters.
Time for G1, S, G2, M
For tumours?
Cell cycle time varies among different tissues
○ Bone marrow and gastrointestinal epithelial cells have short cell cycle times
● Time for G2, S and M phases are similar for different cell types
● G1 is the most variable, ranging from 1-2 hours to months.
● S~ 6 hrs
● G2~1-3 hrs
● M~1-2 hrs
Cell cycle time for tumours are similar to normal cells
○ SCC ~ 30-40 hours.
Compare DNA damge in G1, S, G2 early and late
Think the more repair mechanisms and the more accessible the DNA the less sensitive (i.e more able to repair)
Most to least sensitive: M>G2late > G2early > G1 > Searly > Slate
Need to know the graph (x axis Gy, Y cell survival)
G1: DNA damage leads to ATM-dependent stabilization and activation of p53 (Also ATR activation to a much lesser degree)
S: intra phase checkpoint delays replication and initiates repair.Reduction in rate of DNA synthesis is dose dependent. Regulated by ATM and ATR
G2 early: Only small doses required to activate the checkpoint. Rapid drop in mitotic cells after irradiation. ● ATM/ATR- CHK1/2-CDC25A/C
G2/M most sensitive to damage
Early G1 and S least sensitive (more access to repair mechanisms)
G2 Late: Indep of ATM, ATR dependent. Long delay which is dose dependent. Likely reflects damage that persists after irradiation in G1 or S phase
Describe how a eukaryote cell nucleus is enclosed
Double membrane enclosed organelle (the nuclear envelope) communication with the cytoplasm is mediated by protein lined nuclear pores
A transcribed segment of a gene is translated into RNA and contains:
Which part is lat a removed and what is then name of the process?
Each end contains?
Introns and exons
Exons are removed by splicing
Each end contains a 5’ and 3’ untranslated region
What is a reading frame?
What is an open reading frame?
Divides the nucleotide sequence into non overlapping nucleotide triplets which specify specific amino acids.
An open reading frame is a continuous sequence of triplets from start codon to stop codon
The most common start codon is?
The most common start codon is AUG. The start codon is often preceded by a 5’ untranslated region (5’ UTR).
Non-transcribed parts of a gene include:
Start and stop codons
Regulatory segments: promotors, silencers that help control expression.
Chemically DNA strands are what type of polymer?
The monomers are?
What do these monomers consist of?
Polynucleotides
Nucleotides:
- Nucleobase (adenine, guanine, cysteine, thymine)
- Deoxyribose sugar group
- Phosphate group
The deoxyribose phosphate group forms the backbone
What are the nucleobase pairs?
They are paired by what type of bonds?
Nucleobase pairs C-G, A-T
With hydrogen bonds
From the level of DNA double helix describe packaging into chromosome:
Differentiate between packing for dividing and non-diving cells
1) Nucleosome: DNA double strand wraps around 8 histones twice (really 1.65 times)
2) Chromatosome: Nucleosomes pair with a H1 histone to form a chromatosome (DNA wrapped twice around the 9 histone configuration (about 11nm wide). Strands now 7 times shorter than fully unravelled DNA.
“Beads on a string” - genes under active transcription
3) 30nm fibre: Nucleosomes coiled into compact 30nm fibre - these are less active genes
During mitosis chromatin is further compacted using scaffold proteins into the classic for-arm chromosome
Basic components of a four arm chromosome:
sister chromatids joined by a centromere
Brief outline of the process of DNA replication:
Why is it semi-conservative?
The process of creating two DNA double stands from a single double strand.
Both strands of Original DNA are used to create complimentary strands.
Semi-conservative because each daughter double strand contains one strand of the original DNA double strand
To begin replication of DNA a special protein (?name) must interact with DNA at a specific site (?name). What next steps does this interaction lead to?
Initiator protein binds to a replication origin
Starts to unwind DNA, attracts the proteins that make up the replication machine
What are the subunits of the replicator machine and their function?
Helicase (+single stranded binding protein) unwind DNA
Gyrate makes nicks to prevent supercoiling
After DNA has been unwound at the replication origin, what is the next step?
Initiator protein bind replicator origin -> helicase + single-stranded binding protein and DNA gyrase recruited.
2 replicator machines built
2 replication forks formed at origin
Machines move away from replication origin, as forks open in opposite directions
As the replication forks open what are the key proteins needed to create a daughter strand from the template?
DNA polymerase, this in tern needs RNA polymerase to generate a 3’ end (primer) for polymerase.
In what direction are DNA strand replicated?
This creates issues because
5’ to 3’ - i.e DNA polymerase adds nucleotides to the 3’ end.
Because DNA replication is directional, what happens at the lagging strand?
as the replication fork moves long the strand extending in the 3’ to 5’ direction needs to be replicated using fragments produced 5’ to 3’ and put together (Okazaki fragments).
The other role of DNA polymerase
Proofreading after each nucleotide added to ensure no mismatch. If error then 3-5’ exonuclase removes
3 basic differences between RNA and DNA:
Ribose rather tha deoxyribose backbone
Uracil instead of thymine
single stranded and oft folded
RNA polymerase binds to …… of the gene.
○ Once bound, the polymerase …… the double helix.
○ One of the DNA strands act as template for RNA synthesis which continues to ………
RNA polymerase binds to promoter region (TATA box) of the gene.
Once bound, the polymerase opens up the double helix.
One of the DNA strands act as template for RNA synthesis which continues to the terminator site (stop site).
RNA is synthesized in the ….. direction.
○ Synthesized RNA is processed to increase stability
Adding what to both ends?
RNA is synthesized in the 5’-3’ direction.
○ Synthesized RNA is processed to increase stability:
■ Capping modifies the 5’ end
■ A poly-A tail is added at the 3’ end
After a 5’ cap and 3’ poly A tail is added what is the final step to make mature mRNA
Exons are spliced and exons stitched together
○ mRNA can be translated in three possible reading frames but only one is used.
How?
Ribosome searches for start codon (usually methionine) to begin translation
mRNA is pulled through the ribosome in the 5’-3’ direction and is read in nucleotide triplets until stop codon reached (e.g UAG)
2 Main factors other than DNA that regulate gene expression
Epigenetic factors:
Methylation - Increased methylation of promotor regions leads to decreased expression
Acetylation - of histones makes DNA more accessible
Acetylation results in
○ Acetylation of histone proteins alter DNA structure, making them more accessible
○ Acetyl groups also attract proteins that promote transcription
○ Acetylation of different histones has different effects:
■ acetylation of p53 causes activation
■ acetylation of BCL-6 (transcription inhibitor) causes inactivation
Direct action of CHK2 (Also CHK1)?
To which pathway (trigger, sensor, transducer, activator) does each belong?
DSB: MRN-ATM-CHK2-p53 SSB: RPA+ssDNA-ATR+ATRIP-TOPBP1-CHK1-p53 Direct action of CHK2: Inhibit CDC25CC which maintains CDK2 (i.e leads to DEphosphorylaed = inactivated CDK2) CHK1 also does this tion and release of E2F)
Broadly the two key pathways to cell arrest are those that respond to DNA damage (name 2) and those that respond to stress (name 2 and name 2 key triggers which are surrogates for persisting cell problems/DNA issues)
1) DNA damage. DSB =MRN-ATM-CHK2, SSB=RPA-ATRIP+ATR-CHK1
2) [Reactive Oxygen Species], mitogen stimulation: The INK4a = p16 - block Cyclin D CDK4/6 complex
ARF = ARF-MDM2-p53-p21 inhibit CyclinD-CDK4/6
INK4a, ARF, INK4b are at the same location on chromosome9
p16 Pathway:
G1:
Increased ROS -> p16 - inhibits cyclin D-CDK4/6 complex (Prevents phosphorylation of Rb to relate E2F)
Extrinsic control of cell growth and proliferation is achieved by what hormone and what class of signalling molecule?
Growth hormone
Growth factors
Growth factor activity is mediated by binding of ?receptors that influence expression of genes that can:
Besides growth, GFs can drive?
Bind Tyrosine kinase receptors:
○ promote entry into cell cycle
○ remove blocks on cell cycle progression
○ prevent apoptosis
○ enhance biosynthesis of cellular components required for cell growth and division.
Can also drive cellular migration, differentiation and synthetic capacity.
Gain of function mutations lead to ……
Gain of function mutations lead to uncontrolled proliferative ability.
The EGFR family includes:
Epidermal Growth Factor and Transforming Growth Factor α
EGF is:
1) Produced by?
2) Has what effect?
3) Targets which cells?
4) Mutations implicated in which tumours?
■ Produced by macrophages and epithelial cells
■ mitogenic for epithelial cells, hepatocytes and fibroblasts.
■ Mutations/amplifications implicated in many tumours: lung, brain, head and neck, breast.
VEGF is:
1) Produced by?
2) Induced by?
3) Stimulates?
■ Produced by mesenchymal cells
■ Induced by hypoxia and other factors (eg. PDGF, TGF-α)
■ stimulates lymph and angiogenesis.
■ VEGF antibodies used in treatment of renal and colon cancers.
Growth Hormone ● synthesized, stored and secreted by? ● regulated by? ● Activates the? ● Also stimulates?
Growth Hormone
● synthesized, stored and secreted by the anterior pituitary gland.
● regulated by the hypothalamus
● Activates the MAPK/ERK pathway, stimulating cell division esp of chondrocytes.
● Also stimulates insulin-like growth factor-1 which activate the PI3K-AKT-MTOR pathway, promoting proliferation and inhibiting apoptosis.
Receptor tyrosine kinases ○ transmembrane proteins with extracellular an ......... domain and intracellular ........... domain. ○ Usually activated transiently by: ○ activated receptor ......... ○ Can be .......... activated in tumours
Receptor tyrosine kinases
○ transmembrane proteins with extracellular ligand-binding domain and intracellular tyrosine kinase domain.
○ Usually activated transiently by binding of specific growth factor, causing dimerisation
○ activated receptor autophosphorylates tyrosine residues and recruits signalling proteins.
○ Can be constitutively activated in tumours
Give 3 ontologically important TKRs:
EGFR: ERBB1 mutation (lung) ERBB2 mutation (breast)
ALK: translocation and point mutation in lung adenoma and lymphomas.
VEGF
Activation of TKR stimulate 3 pathways:
The ultimate end-point of this is?
These are all frequently involved in what type of mutation?
RTK is the initial event for all 1) RAS (most important to remember, i.e. Cyclin D-CDK4 trigger, also stimulates MAPK) 2) MAPK aka the RTK-RAS-MET-ERK pathway 3) PI3K/AKT: RTK-AKT-mTOR The end-point is cell proliferation
All frequently involved in gain of function mutations
How is RAS activated?
Primary protein for reversing this?
Growth factor binds RTK activated RTK tyrosine kinase phosphorylates RAS-GDP to RAS-GTP
Inactivated by the GTPase neurofibromin
Give the PI3K pathway
What inhibits it?
Draw a diagram if possible
RTK-PI3K-AKT-mTOR promotion of cyclin D (ie. G1 to S)
Inhibited by PTEN (i.e a tumour suppressor gene)
● AKT also inactivates BAD, a pro-apoptotic protein
○ PTEN mutations seen in endometrial cancer
● PI3K activity inhibited by PTEN (hence tumour suppressor).
○ PI3K mutation seen in breast cancers.
Give the MAPK (MAPKinase) Pathway
Most well-known mutation
RTK binding -> RAS-RAF-MEK-ERK-transcription
BRAF mutation in hairy cell leukemias, melanomas, colon cancers
EGF binds what type of receptor?
What is the classic cancer in which over expression is seen?
Overexpression of EGFR, through either mutation or amplification is implicated in several cancers. 2 examples are?
Tyrosine kinase
Over expression of EGF is seen in pancreatic cancers, and is associated with more aggressive phenotype and poorer survival.
Overexpression of EGFR, through mutation or amplification is implicated in several cancers eg. lung and breast.
Hallmarks of Cancer - 6+2?
6 Hallmarks + 2 enabling characteristics
1) Sustained proliferative signaling (e.g. autocrine, increased TKR or GFs)
2) Evading Growth Suppressors
3) Resisting Cell Death
4) Angiogenesis
5) Activating invasion and/or metastasis
6) Replicative immortality
Enablers:
Genome Instability
Tumour-promoting inflammation
Emerging hallmarks:
Evading immune destruction
Deregulating cellular energetics
Arguably the most fundamental hallmark of cancer:
By what pathways does it achieve this?
Sustaining proliferative signalling:
Increase receptors
Mutate receptors to be more active
Increase GFs or stimulate other cells to release
Autocrine activation
Constitutive activation of a component of signalling pathway
Give an example of GF over expression (include events at the receptor, and in what diseases it may be seen):
Overexpression of EGF
● Produced by macrophages and epithelial cells
● EGF binds to EGFR which dimerizes and phosphorylates its tyrosine kinase residues, activating pathways that lead to cellular proliferation
● Hence EGF is mitogenic.
● Overexpression of EGF is seen in pancreatic cancers, and is associated with more aggressive phenotype and poorer survival.
The two main types of mutations that may lead to autonomous functioning of signal transduction pathways:
Give examples of each
Signal transduction pathways are chain reactions of activated intracellular molecules leading to cellular proliferation
Gain of function:
● Most relevant are Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways.
● Loss of function of inhibitory molecules eg. GAP and PTEN also lead to oncogenesis
High risk subtypes HPV
High risk subtypes HPV are 16 and 18
Broadly HPV mediates what, which leads to cancer
HPV mediates loss of tumour suppressor function
All HPV are non-enveloped ……….-………… …… viruses.
Encode X major proteins, Y located in the “early” region Z in the “late” region
All HPV are non-enveloped double stranded DNA viruses.
Encode eight major proteins, 6 located in the “early” (i.e. E1,..E6) region 2 in the “late” region
What are the broad classes of protein coded for by the HPV genome
They are in how many key open reading frames?
Late encode capsid Replication proteins ONCOGENES - E5,6,7 ○ L1,2 encode capsid proteins ○ E1,2,4 regulate viral replication ○ E 5,6,7 are oncogenes.
What are the HPV oncogenenes:
How are they expressed?
E5, E6, E7
HPC invades basal layer of epithelium which acts as a continuous reservoir of HPV DNA.
Inserts into DNA (i.e it is DS-DNA virus), host expresses oncoproteins
HPV E5 oncoprotein does what?
(5,6,7 EGF, p53, Rb)
Activates EGF signalling pathway (i.e RAS-Raf-Mek-Erk, and PI3K-AKT-mTOR) ->Cell growth and proliferation
HPV E6 oncoprotein does what?
(5,6,7 EGF, p53, Rb)
Binds p53 induces its degredation
HPV E7 oncoprotein does what?
(5,6,7 EGF, p53, Rb)
Binds Rb releasing E2F: Cyclin E increases, Cylcin A-CDK2 stimulated, cell goes into S Phase.
Inactivates CIP/KIP (p21, p27)
Broadly the HPV open reading frame segments do what?
ORFs E5,6 &7
Promote check point inhibition and mitosis
Define: Oncogene
Mutated genes that encode proteins which result in continuous replicative potential of the cell
Define: Tumour suppressor gene
Genes that check and regulate cell growth and repair, preventing proliferation of mutated or damaged cells.
Name 4 mechanisms for the activation of oncogenes and/or loss of function of tumour suppressor genes:
1) Point mutation = change of a single base
2) Deletions and insertions: single or pair of BASE PAIRS deleted = frameshift mutations.
IFF pairs involved are in multiples of 3 then an abnormal protein results - either gaining or loosing an amino acid
3) Translocations = re-arrangement of sequences between non-homologous chromosomes
4) Amplifications: Overexpression due to reduplication and presence of multiple copies within the cell. Can be double minutes (extrachromosomal) or homog eneously staining regions (intrachromosomal).
Deletion mutations often result in?
2 key examples are?
Deletions usually result in loss of tumour suppressor genes eg. Rb1 and VHL genes.
What are translocation mutations?
Rearrangement of parts between nonhomologous chromosomes.
What are proto-oncogenes?
How are they activated
Proto-oncogenes are translocation mutations.
Activated by loss of regulatory element or gain of promotor:
○ removal from their regulatory elements eg. Burkitt’s Lymphoma t(8,14)
○ formation of hybrid oncogenes that encode growth promoting oncoproteins. CML t(9,22)
What are amplification mutations?
Give an example
Overexpression due to reduplication and presence of multiple copies within the cell. Can be double minutes (extrachromosomal) or homogeneously staining regions (intrachromosomal).
Eg. ERBB2 (HER2 receptor) in breast, ovarian and stomach cancer, NMYC in neuroblastoma, Cyclin D1 gene in head and neck cancers.
How are oncogenes and tumour suppressor gene mutations quantified?
What is the general principle of both
Either:
Comparative Genome Hybridisation (used to detect of chromosomal copy number changes)
In-Situ Hybridisation
Both methods use DNA or RNA probe(s) labelled with fluorescent dye.
What is Comparative Genome Hybridisation?
What is it used for?
Detection of chromosomal copy number changes
○ Test DNA and reference (normal) DNA are labelled with different fluorescent dyes.
○ The samples are hybridized to an array with DNA probes spanning all 22 autosomes and sex chromosomes.
○ The binding of the samples are compared
○ If both samples bind equally the spots will fluoresce yellow
○ If deletion or duplication is present, fluorescence will skew towards red or green
What is In-situ Hybridisation?
Technique for identifying/quantifying an RNA/DNA sequence of interest using RNA/DNA probes labelled with a fluorescent dye (FISH) that hybridise to a target sequence.
What are the steps in In-Situ Hybridisation?
○ DNA denaturation by heating, separates the strands.
○ Probes are introduced, labelled with fluorescent dye (FISH).
○ Probes are hybridized to target sequence
○ Sample is washed, removing excess, unbound probes.
○ Sample is viewed under fluorescent microscope
○ Spectral karyotyping (multicolour FISH) uses different coloured fluorescent labels to visualize the entire genome.
Chromosomal structural aberrations are divided into main types? 2 examples of each?
How do they differ in outcomes?
Chromosomal structural aberrations are either:
1) Stable (there are for): Are not lethal to the cell and include translocations and deletions (as well as duplications and inversions)
2) Unstable: Gross changes to chromosomes that are lethal to cells - e.g Dicentrics (anaphase bridge), ring chromosomes
What is Knudson’s 2 hit hypothesis?
● Model of tumour suppressor gene inactivation by Knudson in familial retinoblastoma.
● Developed bilateral disease and much earlier.
● Proposed that patients inherited a germline mutation in one RB gene but required a mutation in the other (‘second hit’) to develop RB, whereas sporadic patients required mutations in both genes.
● Thus, tumour suppressor genes are recessive genes that require inactivation of both functional copies before malignancy develops, whereas loss of one functional copy leads to increased susceptibility.
Knudson’s 2 hit hypothesis relates to what type of genes?
What does it imply about those genes?
How does it explain early presentation of a disease?
Tumour suppressor genes
Tumour suppressor genes are recessive genes that require inactivation of both functional copies before malignancy develops
Infant patients inherited a germline mutation in one Rb gene but required a mutation in the other (‘second hit’) to develop RB, whereas sporadic patients required mutations in both genes.
As one mutant is inherited (leaving only one functional allele) early (infant) disease has an autosomal dominant pattern.
Factors other than DNA sequences that regulate (onco)gene expression?
Epigenetic changes.
1) Methylation - Decrease access to DNA. Hyper and hypomethylation seen in many tumours, leading to under or over production of multiple genes.
2) Acetylation - Increase access to DNA. Acetyl groups attract promotors
● Altered DNA methylation throughout the genome is seen in various tumours, as ………. or ………methylation
● Hypomethylated genomes also lead to?
● Local hypermethylation of the promoter regions of tumour suppressor genes lead?
● Usually hypermethylation occurs in one allele, the other functional copy?
● One example?
● Altered DNA methylation throughout the genome is seen in various tumours, as hyper or hypomethylation
● Hypomethylated genomes also lead to chromosomal instability.
● Local hypermethylation of the promoter regions of tumour suppressor genes lead to silencing.
● Usually hypermethylation occurs in one allele, and the other functional copy is lost through another mechanism eg. deletion or point mutation.
● One example is CDKN2A which is a locus that encodes tumour suppressors p14 and p16 that enhance p53 and pRb activity.
CREBBP (a histone acetyltransferase) mutation is seen in 40% of?
CREBBP (a histone acetyltransferase) mutation is seen in 40% of DLBCL.
Carcinogenesis involves which 2 complimentary processes?
Briefly describe each:
Carcinogenesis involves Initiation and Promotion
● Initiation results from exposure to carcinogens causing permanent DNA damage.
● Promoters are non-tumourigenic but enhance the proliferation of initiated cells. Eg. unopposed oestrogen and chronic inflammation.
What are ‘ultimate carcinogens’
Some carcinogens require metabolic activation into
‘ultimate carcinogens’
Vogelstein model?
Vogelstein model of colon adenocarcinoma- the adenoma-carcinoma sequence:
Hyperplasia (1→ Dysplasia (2→ Adenoma (3→ Carcinoma
1) APC - Adenomatous Polyposis Coli
2) K-Ras - GTPase mutation leading to increased proliferative signal
3) p53 - decreased apoptosis and cell cycle arrest
Studies of tumour gene expression do not support this model (e.g. data sows very rare to have both K-Ras and p53 mutation)
2 Main cell types (beside control!!) used for in-vitro models of tumours?
1) Transformed cells are created by irradiation of embryo or fibroblast cells.
2) Malignant cells
Compare the appearance of transformed and malignant cells when plated:
What are the experimental benefits of Malignant cells?
Major pitfall?
● Transformed cells are created by irradiation of embryo or fibroblast cells.
○ When plated, cells replicate in disorganized fashion
● Malignant cells
○ when grown in suspension grow as spheroids = large spheric clump of cells
○ The spheroids mimic characteristics of solid tumours, with hypoxic core and cycling cells on the outside
○ They are reproducible and inexpensive
○ However they lack ECM and vasculature, hence not a fully accurate model of tumour behaviour.
5 key histological difference between benign and malignant cells (a common exam question is to make a table):
Benign:
1) Well Differentiated
2) Few mitotic figures, appear normal
3) Normal Karyotype
4) Limited number of cell divisions
5) Exhibits contact inhibition- Stops proliferating when cells come into contact with each other
Malignant:
1) Well to poorly differentiated
2) Many mitotic figures which appear bizarre
3) Abnormal karyotype: Abnormal number of chromosomes or chromosomes with abnormal structure
4) May proliferate indefinitely in culture
Can produce telomerase
5) No contact inhibition- will continue to pile up into mounds of cells
Under normal conditions, Neovascularization is triggered by?
Broadly after being triggered what is activated and where do they go? :
Neovascularization :
○ Injury of local basement membrane or hypoxia
○ Pro-angiogenic factors activate endothelial cells which migrate to the site of tissue injury
Steps of normal neovascularisation:
Tumour angiogeneis often leads to subobtimal angiogenesis at which phase?
1) Pro-angiogenic factors activate endothelial cells which migrate to the site of tissue injury
2) Endothelial cell proliferation
3) Regulated by angiogenic factors
4) After neovascularization there is a resolution phase where blood vessels mature and stabilize
■ This phase occurs less in tumour angiogenesis hence tumour blood vessels are crap (leaky, disorganised, small, prone to collapse)
After angiogenesis there is a resolution phase where blood vessels mature and stabilize
■ This phase occurs ……… in tumour angiogenesis
■ Tumour blood vessels are:
After angiogenesis there is a resolution phase where blood vessels mature and stabilize
■ This phase occurs less in tumour angiogenesis hence tumour blood vessels are:
● leaky ● disorganized ● smaller in diameter ● prone to collapse ● high permeability to large molecules ● variable blood flow
Angiogenesis is controlled by balance between?
Examples of each?
Catch phrase to describe this
Angiogenesis is controlled by balance between pro- and antiangiogenic factors. When the balance is in favour of angiogenesis “angiogenic switch”
○ pro: bFGF (FGF-2), VEGF, TGF alpha and beta, TNF alpha
○ anti: angiostatin, endostatin, thrombospondin-1, Interferon
Targets for regulation of tumour angiogenesis include:
● Regulators include proteases (produced by the tumour or ECM), p53, RAS/MAPK signalling
● Hypoxia is a major trigger of angiogenesis.
● VEGF inhibitors not as effective as hoped suggesting other escape pathways.
8 Steps of Metastatic Cascade:
1) Dissociation of cancer cells from each other
Loss of E-cadherin facilitates detachment and infiltration of surrounding tissues. E.g. SNAIL and TWIST downregulate expression.
2) Degradation of basement membrane
Secretion of proteases. Changes in attachment to ECM proteins.
3) This enables Locomotion
4) Movement through the basement membrane
and Intravasation = Invasion of endothelium to enter the circulation
5) Formation of tumour emboli
Tumour cells aggregate in clumps by adhesion with platelets and T-lymphocytes
6) Adhesion to endothelium basement membrane and extravasation
Adhesion molecules involved: CD44 adhesion molecule expressed on normal T lymphocytes
7) Arrest at distant organ site, Trapping in first capillary bed, tumour organ topism
8) Survival in tumour microenvironment
e.g. Angiogenesis, evasion of immune mediated destruction
9) Colony formation
Intravascularly, tumour cells are vulnerable to destruction in 3 ways:
Intravascularly, tumour cells are vulnerable to destruction
● mechanical shear stress
● apoptosis
● innate and adaptive immunity
SNAIL and TWIST downregulate expression of?
Why is this important?
1 step of Metastatic cascade.
Dissociation of cancer cells from each other
Loss of E-cadherin facilitates detachment and infiltration of surrounding tissues. E.g. SNAIL and TWIST downregulate expression.
Numerically tumour growth typically exhibits:
Sigmoid (Gompertz) growth.
Where the mass is typically only clinically detectable at the plateau phase.
Sigmoid Characterised by:
1) Initial Exponential Growth: Small number of tumour cells with adequate nutrition undergo division. Each progeny is able to survive and divide. The growth fraction close to 100%.
2) Linear Growth: Competition for nutrients/oxygen make tumour micro environment less favourable, not all progeny survive.
3) Plateau: Tumour microenvironment increasingly unfavourable - only a reducing minority of cells are able to proliferate. Eventuallycell loss factor is equal to the growth fraction. The tumour mass is clinically detectable and causing symptoms. May also be areas of necrosis from hypoxia.
In drawing a Gompertz curve for tumour growth how would you label the x and y axis?
Y - axis: Number of cells 10^9 [0 .2, 0.4,….. 1]
X Axis: Time (Arbitrary units)
Define Growth Fraction in terms of tumour growth
What is it’s opposite (i.e the opposite force acting especially in the Gompertzian plateau)?
The ratio of proliferating cells over total number of cells
Cell loss factor: The ratio of the rate of cell loss over the rate of new cell proliferation
Tumour doubling time is the:
Potential doubling time:
What is Pdt useful for?
Tumour doubling time is the time taken for a tumour to double its initial volume.
Potential doubling time is the theoretical doubling time in the absence of cell loss. Used to calculate cell loss Fraction.
Tpot = Tc/GF (Tc = cell cycle time, GF = growth fraction)
i.e (time to make new cell/number of proliferating cells) = number of new cells per unit time (assuming no death) (e.g. cells/sec)
Define Cell loss factor:
The ratio of the rate of cell loss over the rate of new cell proliferation
● 1- (Tpot/Td) where Tpot is the potential doubling time and Td is the actual doubling time.
Determinants of tumour growth RATE:
1) Cell cycle time
2) Growth Fraction (#prolif/#cells)
3) Cell Loss Factor = 1- (potDt/Dt)
4) Accelerated re-population
5) Tumour micro environment: stroma and hypoxia
Hypoxia activates pathways that allow tumour cells to adapt to hypoxic stress. 3 Key adaptations:
Hypoxia activates pathways that allow tumour cells to adapt to hypoxic stress. 3 Key adaptations:
○ anaerobic glycolysis
○ changes in blood flow
○ stimulate angiogenesis.
The concept that tumour doubling time for certain tumours is shorter during or shortly after a course of treatment compared to an untreated tumour = ?
In experiments of SCC head and neck, this seems to take place approximately:
Accelerated re-population
In experiments of SCC head and neck, this seems to take place approximately 28 days after starting treatment.
● The dose required to compensate for this repopulation is about 0.6Gy/day
2 key components thought to underlie accelerated re-population:
1) Improved/less competitive tumour microenvironment: reduction in number of tumour cells, improvement in oxygen and nutrient supply to the remaining tumour cells resulting in a more favourable tumour microenvironment.
2) regeneration response of clonogenic cells, similar to that seen in normal epithelial tissue, potentially triggered by EGFR
Approaches to counteract Accelerated Repopulation:
Methods to counteract this is to reduce overall treatment time by accelerated hyperfractionated treatment, combined chemoradiotherapy, or higher total dose.
Tumours become detectable at about X cells?
Tumours become detectable at about 10^9 cells
Earliest event in sensing DNA damage?
Phosphorylation of H2AX to gammaH2AX
H2AX → γH2AX
Leads to:
Can be seen as
Leads to recruitment of proteins to site of DNA damage
Form ionising radiation induced foci (IRIF) - if stained with antibodies.
3 related kinases can phosphorylate H2AX at sites of DNA damage:
3 related kinases can phosphorylate H2AX at sites of DNA damage:
1) ATM-MRN (the main one)
2) DNA-PKcs-KU
3) ATR-ATRIP
The main sensor pathway for DNA damage?
What are the steps?
■ MRN (MRE11, RAD50, NBS1) assembles at site of DNA breaks
■ MRN recruits and activates ATM
■ ATM phosphorylates H2AX
Potential doubling time?
Potential doubling time:
Tumour doubling time when there is no cell loss
Tpot = Tc/GF (Tc = cell cycle time, GF = growth fraction)
Cell loss factor:
Cell loss factor:
Ratio of rate of cell loss to rate of new cell production
1 – Tpot/VDT
What is volume doubling time be dependent on?
Number of cells replicating and time for them to replicate is dependant on:
1) Number of tumour cells actively going through cell cycle and replicating (growth fraction)
2) Duration of cell cycle in these cells (cell cycle time)
3) Rate of Cell Loss
Define growth fraction:
Number of Cells Replicating/number of cells in tumour
Lab method for determining the ratio of replicating cells to total number of cells (aka?)
Ki67 proliferation index in a tumour is an Indication of growth fraction:
Ki67 cell cycle specific protein
Detected by IHC using monoclonal antibody
Most solid tumours have growth fractions below:
Most solid tumours have growth fractions below 50%
How long is an average cell cycle?
For a typical rapidly proliferating human cell with a total cycle time of 24 hours, the G1 phase might last about 11 hours, S phase about 8 hours, G2 about 4 hours, and M about 1 hour.
Common biochemical technique for studying cell cycle kinetics
2 Examples:
1) Identification of S phase cells by incorporation of radioactive thymidine.
2) Cells at different stages of the cell cycle can also be distinguished by their DNA content. Cells in G1 are diploid so their DNA content is referred to as 2n. Cells in S have DNA contents ranging from 2n to 4n. DNA content remains at 4n for cells in G2 and M, decreasing to 2n after cytokinesis.
Incubation of cells with a fluorescent dye that binds to DNA, followed by analysis of the fluorescence intensity of individual cells in a flow cytometer
For 1 Gy of radiation
>? damaged bases
about ? ss DNA breaks
about ? ds DNA breaks
For 1 Gy of radiation
>1000 damaged bases
about 500 - 1000 ss DNA breaks
about 20 - 40 ds DNA breaks
Repair mechanism for each type of IR induced DNA damage:
● Damage to bases:
● Single strand breaks:
● Double stranded breaks:
Types of DNA damage not seen in therapeutic range IR
● Damage to bases- repaired through base excision repair or nucleotide excision repair
● Single strand breaks- repaired through SSB repair
● Double stranded breaks- repaired through homologous recombination or non-homologous end-joining.
● Other lesions not directly related to radiation damage:
○ mismatch bases can occur when attempting replication- repaired through mismatch repair
○ bulky lesions or DNA adducts formed by UV light or chemotherapy- repaired through nucleotide excision repair.
What is histone H2AX (in relation to there histones)?
Variant of histone H2A
10 – 15% total cellular histone H2A
Distributed throughout nucleus
H2AX phosphorylation occurs at site of DNA damage, within 30 mins of DNA damage
Recruits proteins to sites of DNA damage and signals activation of effector pathways
ataxia–telangiectasia patients have a mutation in?
Which causes?
ATM mutation: H2AX-MRN-ATM-CHK2
Sufferers of AT or cells that have inhibition of ATM are very radiosensitive – defective DNA damage response
What does H2AX do?
What is the key activator?
Much lesser influence?
When phosphorylated to gammaH2AX recruits proteins to sites of DNA damage and signals activation of effector pathways.
MRN-ATM is key activator (missing in AT patients), if missing ATR-ATRIP and DNA-PKcs-Ku will phosphorylate H2AX
2 Main mechanisms for DSB repair.
For the error-free mechanism, what is needed for this to occur.
Homologous recombination - slow, but error free
Non-homologous end-joining - Fast, cell can survive DSB, but deletion and insertion errors.
For Homologous recombination to occur there bust be a sister chromatid therefore DSB must occur in late S and G2.
Steps in Homologous recombination
Homologous Recombination uses sister DNA with the same sequence as a template for repair:
1) Single stranded filaments made around DSB
2) Filaments coated with RPA
3) RAD51 displaces RPA and SEARCHES and INVADES sister chromatid
4) Helices UNWIND and polymerases SYNTHESISE
5) Finally crossover points are CUT by RESOLVES, and ligated.
Steps in Non-homologous End-joining:
Detection→Recruitment/activation→Processing→Ligation:
1) Detection: DSBs detected by Ku70 and Ku80 proteins which bind to site. They protect ends of DNA strands, prevent exonucleases degrading DNA.
2) Recruit: DNA-PKcs recruited - autophosphorylates & phosphorylates other proteins.
Artemis (protein complex) recruited to DNA break, forms complex with DNA-PKcs and is activated by phosphorylation.
3) Processing: Artemis endonuclease activity processes the DNA ends ready for ligation. If DNA damage or repair process causes a non-blunt end of DNA i.e. a 3’ or 5’ overhang, then the absent DNA can be generated by polymerases.
4) DNA ends ligated through ligase IV and XRRC4 and XLF proteins.
In the Processing phase of NHEJ:
……… ……….. activity processes the DNA ends ready for ligation.
If DNA damage or repair process causes a non-……. end of DNA i.e. a 3’ or 5’ ………, then the absent DNA can be generated by ………….
Artemis endonuclease activity processes the DNA ends ready for ligation.
If DNA damage or repair process causes a non-blunt end of DNA i.e. a 3’ or 5’ overhang, then the absent DNA can be generated by polymerases.
Mutations in genes associated with which four DNA repair mechanisms are associated with increased radio sensitivity?
What type of DNA damage is associated with each?
1) Damage to Base = Base excision and repair
2) SSB = Single Stranded Brake Repair
3) DSB - Non-homologous End joining
4) DSB - Homologous Repair
Basic steps of Base excision repair:
Damaged DNA detected and removed by glycosylases:
○ Each glycosylase is specific for a particular type of base damage.
○ They cut out the damaged base without cutting the DNA backbone resulting in an abasic site.
2) AP endonuclease then cuts the DNA backbone making a nick (SSB).
3) Subsequent repairs follow one of two pathways:
■ Short patch
● involves replacing the damaged base only
● DNA synthesis carried out by DNA polymerase β
● Ligated by ligase 3
■ Long patch
● involves cutting out and replacing up to 10 nucleotides.
● DNA polymerases δ and ε
● ligase 1
Basic steps of Single Stranded Break Repair
1) Damage detected by PARP-1.
2) Breaks are ‘dirty’ and require end-processing step by polynucleotide kinase PNK
BER (ie. proceeds from the point of nick):
3)Once clean nick produced, short or long patch repair follows BER.
■ Short patch
● involves replacing the damaged base only
● DNA synthesis carried out by DNA polymerase β
● Ligated by ligase 3
■ Long patch
● involves cutting out and replacing up to 10 nucleotides.
● DNA polymerases δ and ε
● ligase 1
Key points for γ-H2AX:
Phosphorylated form γ-H2AX:
○ recruits proteins involved in DSB repair
○ opens tertiary structure of DNA allowing access to repair proteins
○ activates checkpoint proteins
○ involved in formation of ionizing radiation induced foci
Why may ATR be activated by the ATM-MRN pathway?
ATR may also be activated by ATM-MRN pathway when processing ds DNA breaks which creates ss DNA
ATR-ATRIP-CHK1
MRN-ATM-CHK2
How is IR induced DNA measured:
1) Chromosome aberrations. Assessed for at 1st metaphase after exposure.
2) Assays for DNA damage
i. γ-H2AX - Radiation induced foci immunofluresence
ii. Comet Assay - i.e the more fragmented the DNA the longer the tail behind the head (nucleus)
iii. Pulse field Gel electrophoresis - post electrification (current applied in diff directions) fragmentation corresponds to DNA damage
iv. Micronucleus assay
v. Plasmid assay
Steps for comet assay:
● Single cell electrophoresis
● Irradiated cells embedded in agarose gel on microscope slide
● Cells lysed using high salt and detergent solution
● DNA electrophoresed
● Fluorescent staining of gel performed with ethidium bromide or propidium iodide which is specific for DNA
● DNA fragments move away from nucleus, forming a ‘comet’ with head formed by the nucleus and tail composed of smaller DNA fragments.
● Comet tail length correlates with DNA damage.
Steps for Pulse field Gel electrophoresis:
Pulse field Gel electrophoresis - post electrification (current applied in diff directions) fragmentation:
● Used to assess DNA DSB and large fragments up to 10 Mbp
● Comet assays not effective in base pairs >50kb
● irradiated cells suspended in agarose gel and poured into moulds
● cells lysed and gel electrophoresis performed
● Current applied switching from different directions: straight across and 60 degrees either side
● Larger pieces will react slower to change in direction
● Over the course of time bands will begin to separate
● Gel is read after staining with fluorescent ethidium bromide
● Fragmentation of DNA correlates with amount of DNA damage.
Modes of Cell Death:
Mitotic catastrophe Necrosis Apoptosis (Type 1 Death) Autophagy (Type 2 Death) “Senescence” = Reproductive Death
How does Mitotic catastrophe occur?
Why is it cell death?
Morphological changes:
Occurs after mitosis because cells have entered mitosis with DNA damage still present. Cells can no longer replicate due to the chromosomal damage = Reproductive death. This form of cell death can trigger other cell death pathways such as apoptosis.
Morphological changes: multinucleated giant cells, chromosome aberrations, dicentric nuclei
How does Necrosis occur?
Morphological changes:
Occurs due to unfavourable growth conditions, e.g change in pH in cellular environment, but also due to insults which damage DNA such as radiation.
Morphological changes: cellular swelling and rupture,
In nucleus – uncondensed chromosomes, micronuclei, clumping of DNA
In Cytoplasm - organelle degeneration, mitochondrial swelling, release of lysosomal enzymes, vacuolation of cytoplasm
What is Apoptosis/normal role?
What leads to it?
How is it relevant to cancer?
Regulated form of cell death.
Essential component of development and in maintaining tissue homeostasis
Triggered from within cell or by external cell signals
Changes in apoptosis control can contribute to cancer development
Morphological appearance of Apoptosis:
1) Chromatin condensation and nuclear fragmentation
2) DNA laddering
3) Cell membrane blebbing
4) Formation of apoptotic bodies (vesicle containing parts of a dying cell) - Prevents leakage of potentially damaging cellular proteins
Compare the morphology of necrosis to Apoptosis:
NECROSIS
Cell body: Cellular Swelling
Chromatin: Does not condense
Cell integrity: Blebbing -> cell becomes leaky
Resolution: Cellular and nuclear lysis causes inflammation
APOPTOSIS Cell body: Shrinks Chromatin: Condenses Cell integrity: Budding Resolution: Buds become apoptotic bodies which are phagocytised without inflammation
The apoptosis pathaway can be broken into which two classes of molecule?
Apotosis proceeds through …….. activation of ………..
Main counter balance molecules to this process?
Sensor molecules = sensor caspases
Involved in initiating apoptosis
Activation of sensor caspase (8 or 9)
Effector molecules = Effector caspases
Subsequent activation of downstream effector caspases (including caspase 3)
Sequential activation of caspases
Usually in inactive form (procaspase)
Inhibitors of apoptosis proteins (IAP)
The 2 signal pathways to apoptosis and their key trigger molecules are:
Extrinsic pathway:
■ sensor caspase 8, activated by binding of extracellular ligand which activates death receptor. Not usually activated by radiation damage.
■ Extracellular ligands include – TNF, TRAIL, FAS.
Intrinsic pathway:
■ sensor caspase 9, activated in cell by presence of cell damage (e.g p53 signal).
■ Balance of Pro and anti-apoptotic factors in cell (around or in mitochondria) will determine if sensor caspase 9 activated.
Detail the Extrinsic pathway for apoptosis:
Extrinsic pathway:
1) sensor caspase 8, activated by binding of extracellular ligand which activates death receptor. Eff
■ Extracellular ligands include – TNF, TRAIL, FAS.
2) Effector caspases activated (Caspase 3, also 6 and 7)
3) Apotosis
Detail the Intrinsic pathway for apoptosis:
Intrinsic pathway:
1) Presence of cell damage. Signalled by p53, PI3K-AKT
■ Pro and anti-apoptotic factors in cell (around or in mitochondria) will determine if sensor caspase 9 activated.
■ Normally anti-apoptotic factors predominate and caspase 9 is inactive, but if cell damage occurs pro-apoptotic factors activated (e.g. by p53) and balance changes in favour of caspase 9 activation.
■ Pro-apoptotic proteins include BAX, BAK, PUMA. BAD from PI3K - AKT activates BAX and BAK ■ Anti-apoptotic: BCL-2
IAP family inhibit Caspase 9 and 3.
2) If balance altered: cytochrome C released from mitochondria into cytoplasm - apoptosome formed
Caspase 9 activated
3) Effector caspases especially 3 (others 6 and 7) mediate apotosis.
What is Autophagy?
How can it be activated?
Key regulatory molecule?
Normal role?
Part of cytoplasm of cell digested to release molecules and energy
Can be activated in response to loss of nutrients or growth factors
Regulated by mTOR kinase (i.e. PI3K-AKT-mTOR)
Can prolong cell survival in adverse conditions
Can also lead to a form of programmed cell death (Type II) that is not caspase dependent
Autophagy acting as a tumour suppressor function – if genes associated with initiation of autophagy lost then increased cancer development in mice studies
Evidence of a role for Autophagy in Cancer?
Autophagy acting as a tumour suppressor function – if genes associated with initiation of autophagy lost then increased cancer development in mice studies
Morphological apperance of type II cell death
Autophagy can lead to a form of programmed cell death (Type II) that is not caspase dependent:
Looks like Apotosis
BUT no: DNA laddering, vesicles no apototic bodies (but still vesicles present)
What is senescence?
Usually associated with?
Can occur prematurely when?
Metabolically active but Reproductive Death - Cell-cycle stopped - “G0”
Usually associated with telomere shortening in aging
DNA damage
Premature senescence can be induced by cellular stress and IR DNA damage (not related to telomere length, activated by other pathways).
Different cell types have different propensity to undergo senescence – common in ………. cells after radiation
Different cell types have different propensity to undergo senescence – common in fibroblast cells after radiation
Morphological appearance of Senescence:
Heterochromatic nuclei
Increased granularity of cytoplasm
Flattening of cytoplasm
Cell Death soon after radiation (i.e. within 7 hours) is due to activation of ……. pathway
Which leads to upregulation of:
DNA Damage Response (DDR)
DDR activation of apoptosis: upregulation of pro-apoptotic proteins and activation of p53
Genes regulating apoptosis can influence radiosensitivity of cell in this situation
