MT1

Question 1

What is the restriction point?

Answer 1

Restriction point: point in the cell cycle that, without passing it, and without input of mitogens and growth factors, will lead the cell to return to G0. Passed this point, it is cell autonomous – division will occur regardless of mitogens/growth factor input.

Question 2

Describe the pathway for the restriction point.

Answer 2

• Mitogens lead to the MAPK cascade, leading to its entrance into the nucleus and promoting the transcription of early response genes – include myc, fos, jun TFs. – These are present in the cell during G0
• These then induce the transcription of delayed response genes – D type cyclins, Cyclin E, G1 cdks, E2F
• E2F production stimulates transcription of late G1 cyclins (cyclin E) and s-phase cdk2
• G1 CCC phosphorylates Rb to release E2F
• E2F upregulates production of S-phase CCC  further phosphorylates Rb
• C-fos and jun upregulate E2F
• Leads to S-phase and cell autonomous division

Question 3

Describe the histological differences between benign and malignant tumours
(margin, local invasion, metastases, resemblance to normal, growth rate, chromosomal complement, mitotic activity, abnormal mitosis, cell death, pleomorphism)

Answer 3

o	Tumour margin
	Benign  often pushing
	Malignant  infiltrative
o	Local invasion
	Benign  never
	Malignant  yes
o	Metastases
	Benign  never
	Malignant  frequent
o	Resemblance to normal
	Benign  good
	Malignant  variability – tends to take on other shapes of cells/new shapes
o	Growth rate
	Benign  slow
	Malignant  rapid
o	Chromosomal complement
	Benign  diploid
	Malignant  aneuploidy common
o	Mitotic activity
	Benign  low
	Malignant  high
o	Abnormal mitosis
	Benign  never
	Malignant  common
o	Cell death
	Benign  rare
	Malignant  frequent (apoptosis and necrosis)
o	Cellular and nuclear pleomorphism (variability in size, shape, and staining of cells/nuclei)
	Benign  no
	Malignant  common

Question 4

What are the mechanisms to make a proto-oncogene an oncogene?

Answer 4

o Mechanisms to make oncogenic – Conversion or activation – 4 mechanisms:

1. Point mutation: results in a constitutively active proteins
   a. Point mutation in regulatory domain leading to relief of inhibition (example)
2. Chromosomal translocation – fusion
   a. Fuses two genes together to produce a hybrid encoding a novel protein – ex: Philadelphia chromosome – BRC-ABL
3. Chromosomal translocation – near active promoter/enhancer elements
   a. Translocation that brings a growth regulatory gene near a more active promoter
   b. Ex: c-myc (chr 8) under control of transcriptional enhances of Ig gene (2, 14, 22)
4. Amplification
   a. Resulting from abnormal DNA replication – leading to amplification of the DNA sequence and overproduction of the protein

Question 5

What is tumour suppressor?

Answer 5

• Tumour suppressor: gene that generally plays a repair or control role in the cell cycle and whose loss can lead to loss of control and perhaps malignancy. Often a negative regulator of cell growth.

Question 6

What is an proto-oncogene.

Answer 6

normal cellular constituent that normally serves a growth promoting role in the cell cycle. Through amplification, mutation or modification, the activity of the protein is increased and becomes oncogenic and can promote tumour formation.

Question 7

What activates p53?

Answer 7

• P53 is activated by:
o Lack of nucletodies, UV radiation (ATR), ionizing radiation (ATM), oncogenic signaling (ARF pathway), hypoxia, transcription blockage

Question 8

What does p53 induce?

Answer 8

c• P53 is the central component of the previous DNA damage checkpoints and can induce:
o Cell cycle arrest/senescence, repair, apoptosis, regulation of angiogenesis and return to proliferation

Question 9

Describe the factors that stabilize/destabilize p53

Answer 9

• P53 is a transcription factor that under normal conditions is very unstable and does not accumulate to sufficient levels to stimulate transcription of its targets (namely p21)
o The activity of p53 is held in check by mdm2
o Mdm2 is normally bound to p53 and will both inhibit its activity and ubiquitinate it, leading to its degradation by the proteasome
o Similarly, plk3 will target p53 for degradation
o Different proteins will stabilize p53 and prevent its degradation (ex: plk2, 4, chk1/2)
Oncogenic signaling

Question 10

What are p53 targets?

Answer 10

• P53 has many targets but p21 is a main one. It will induce transcription of p21 to lead to cell cycle arrest in G1, S-phase entry, S-phase and M-phase entry.
• P53 also induces transcription of mdm2  negative regulation

Question 11

Describe oncogenic signaling

Answer 11

• Oncogenic signaling:
o Excessive levels of myc, Ras, etc  result in E2F production  upregulates ARf  associates with mdm2 and drags it to the nucleolus  p53 free from inhibition  promotes apoptosis, cell cycle arrest or repair

Question 12

Describe missense mutations in p53

Answer 12

• P53 is a tetramer
o A missense mutation in one allele will affect the entire structure such that the tetramer contains a defective subunit
 Act as dominant negatives mutations causing loss of function

Question 13

Describe knudson’s model of carcinogenesis.

What are supporting examples?

Answer 13

• Knudson’s model of carcinogenesis:
• At least two independent mutations are needed before tumours can develop
• In the case of familial cancer predisposition, the first mutation is present in the germ cells and thus inherited by every cell
• Initiation requires a second mutation in the same gene on the homologous chromosome
• For sporadic cases, mutations on homologous chromosomes are required in the same cell
• Follows an dominant inheritance pattern for the familial case
o Although there was a dominant inheritance pattern, we are looking at recessive alleles
• Model regarding tumour suppressors
Rb, APC - FAP, LPS

Question 14

Describe clonality.

What are examples?

Answer 14

• Neoplastic tumours are often heterogeneous and contain more than one cell type, but their initiation and continued growth is dependent on a single population of neoplastic cells. These cells are presumably clonal – i.e. derived from the same cell – and will carry the same genetic or epigenetic mutation.
o In this model, cancers evolve by a reiterative process of clonal expansion (i.e. all descendants of a cell will have the same anomaly) and clonal selection – whereby cells with increased proliferative potential are selected and make up future cell populations.

Mosaic X-chromosome - G6PD
G6P-ts
Philadelphia

Question 15

Describe CIN

Answer 15

• Chromosomal instability – CIN – low mutation rate, aneuploid, cant underog repair
o Type of genomic instability in which the chromosomes themselves, in whole or in part, are unstable.

Question 16

Describe MIN

Answer 16

• MIN – microsatellite instability – MIN – repair deficient, typically diploid
o Type of genomic instability that results from impaired DNA mismatch repair (MMR). Cells with abnormal MMR are unable to correct errors occurring during DNA replication which can lead to novel microsatellite fragments.

Question 17

Describe CIMP

Answer 17

• CPG island methylation phenotype – CIMP – epigenetic instability
o Particularly important for genes like Rb
o Source of genomic instability caused by methylation of the promoter of certain genes, most often tumour suppressors leading to development of malignancy
 DNA hypermethylation in CPG rich promoters
o Increased in DMTs are quite common

Question 18

Describe a nucleation assay.

Answer 18

• Nucleation assay
o Ex: Have a cell line and want to test whether it is CIN or MIN
o Culture cells at 37°C
o Move to 4°C  depolymerize all MTs
o Bring back to 37°C for 10-20 seconds
 If the centrosome is present, and functional – will nucleate MTs
o Fix cells with PFA
o Use antibodies against centrosome components – gamma tubulin or centrin
o Use a secondary antibody conjugated to a fluorophore
o If there is a centrosome nucleating mts  source of CIN
o Confirm by imagery

Question 19

Contract a normal cell, immortalized cell and cancer cell

Answer 19

• Normal cell
o Undergoes approx. 50 cell divisions until senescence (telomere shortening)
o Contact inhibition
o Anchorage dependence
o Requires growth factors for division
• Immortalized cell
o Tend to be aneuploidy
o Might lose characteristics of original cell type
o Immortal – produce telomerase
o Display contact inhibition
• Cancer cell
o Immortal – can divide forever (produces telomerase)
o No contact inhibition
o Anchorage independence
o Reduced requirement for growth factors/mitogens
o Tumorigenic – can introduce into an animal model and it will produce tumours
o Tend to lose actin microfilaments
o Increased glucose transport

Question 20

Describe base excision repair.

Answer 20

• Base excision repair
o Used for mismatches – for example a T:G pair
o Number of different glycosylases recognize these mismatches and bind to site
o APEI endonuclease specific for the mutated base is recruited and cuts around the site, removing the base
o App lyase will cut the DNA backbone
o DNA polymerase fills in the excised space
o DNA ligase fills

Question 21

Describe mismatch repair.

Answer 21

•	Mismatch repair
o	Ex: AC pair mismatch
o	MSH2 and MSH6 complex bind adjacent to mismatch and trigger MLH1 endonuclease and PMS2
o	DNA gets cut
o	Helicase unwinds the DNA
o	Exonuclease removes bases
o	DNA polymerase fills in the area with the correct bases
o	Ligase seals it up

Question 22

Describe NER

Answer 22

• Nucleotide excision repair
o Use for mutations, such as thymine dimers caused by UV radiation, that lead to DNA distortion
o This distortion is recognized by XPC and 23B which bind to the region
o These then recruit TF2H helicase which unwinds the DNA in that region
o End up with a bubble of around 25 bases
o Recruit XPF and XPG endonucleases which cut the DNA on either side of the bubble resulting in a 24-32 base gap
o Damaged DNA is released and degraded
o DNA polymerase fills in the region and DNA ligase seals the nicks

Question 23

Describe HR

Answer 23

• Homologous recombination
o Only occurs for double stranded breaks in DNA during S-phase – where a sister chromatid is present
o Double stranded break activates ATM
o ATM recruits RPA, Rad52, BRCA1, BRCA2, NSB1, MRE11 and Rad 50 – exonucleases and other proteins
o These remove a number of nucleotides at the break site
o Result in 3’ overhangs to which Rad51 binds to and forms nuclear-protein filaments
o These nuclear protein filaments invade and base pair with the other allele by Watson and Crick base pairing – forming a double holiday junction
o DNA polymerase extends the base paired region to produce extended 3’ regions
o This region then pairs with the homologous region of the original broken strand
o Remaining gaps are filled with DNA polymerase and ligated by DNA ligase
o Error Free
o BRCA1/2, Mre11 and ATM are crucial to this pathway

Question 24

Describe NHEJ

Answer 24

• Non-homologous end joining
o Used when a double stranded break occurs outside of S-phase
o DNA Pk and KU80/70 heterodimers form a complex on either side of the break
o These complexes bind to the break and form a synapse
o DNA ligated by DNA ligase
o End up with a deletion
o Predominant form of double stranded DNA break repair that introduces mutations

Question 25

Describe the multi-hit theory of cancer induction.

What are supporting examples

Answer 25

o Oncogenic transformation of 3t3 illustrates that multiple mutations are required to convert a normal body cell to a malignant one
o Cancers arise by clonal selection
 Mutation provides a slight growth advantage
o Progeny of mutated cell undergoes another mutation allowing its descendants to grow uncontrollobaly and form a tumour
o A third mutation allows it to outgrow others
o An additional mutation would allow progeny to escape into the blood and establish daughter colonies at other sites

3t3 - plates, crossed
transgenic mice - myc, ras, cross
APC –> polyps (APC), adenomas (Ras), DCC mutation –> (p53) carcinoma

Question 26

Describe the concept of aging and cancer

Answer 26

• How that fits into concept of aging
o Cancer should increase with age because it takes decades for the multiple mutations to occur
o This model suggests a need for 4 mutations and thus we would expect to see an increase in cancer incidence with age

Question 27

Describe cancer stem cell theory.

Answer 27

• Model that posits that not all cells within a tumour are biologically equivalent.
• Stem cells, located at the apex of cell formation, can produce two cell types by asymmetric division: transit amplifying cells and self-renewing stem cells.
• Stem cells can themselves become cancerous
• These two cell types differ in population numbers in a tumour: Transit amplifying cells correspond to the partially differentiated cell types that make up the majority of the tumour and do not replicate with high efficiency.
o Cancer stem cells are the minority population.
o Cancer stem cells are stem cells that have undergone a mutation. All descendants of this cell type will have this mutation. Then, following the principles of clonal expansion from the previous model, mutation after mutation can occur leading to generation of cancer through increased proliferative potential.
• The important thing to keep in mind is that this theory is only an extension of both clonal and multi hit theories
o The CSCs still require multiple mutations or hits to truly become tumourigenic and, many of the cells become clones of one another in the tumour – although this is more of a polyclonal example

Question 28

Describe cancer stem cell theory.

Answer 28

• Model that posits that not all cells within a tumour are biologically equivalent.
• Stem cells, located at the apex of cell formation, can produce two cell types by asymmetric division: transit amplifying cells and self-renewing stem cells.
• Stem cells can themselves become cancerous
• These two cell types differ in population numbers in a tumour: Transit amplifying cells correspond to the partially differentiated cell types that make up the majority of the tumour and do not replicate with high efficiency.
o Cancer stem cells are the minority population.
o Cancer stem cells are stem cells that have undergone a mutation. All descendants of this cell type will have this mutation. Then, following the principles of clonal expansion from the previous model, mutation after mutation can occur leading to generation of cancer through increased proliferative potential.
• The important thing to keep in mind is that this theory is only an extension of both clonal and multi hit theories
o The CSCs still require multiple mutations or hits to truly become tumourigenic and, many of the cells become clones of one another in the tumour – although this is more of a polyclonal example

Question 29

Why are CSCs a problem?

Answer 29

o CSCs provide some inherent issues in treating cancers, commonly for drug therapy
o Example: Gleevec is a common cancer treatment that inhibits may oncogenic proteins such as BCR/ABL, PDGF, KIT receptor, etc.
 It is effective at killing transit amplifying cells however.
 The CSC cells however, tend to become very efficient and develop MDR (multiple drug resistance) pumps
 So, although a patient might respond well to Gleevec, taking the patient off of the drug will lead to cancer return
o To further complicate the issue, if we manage to wipe out the CSCs, these can come back since, these have formed partially differentiated cell types, which can return and form new cancer stem cells – a huge problem

Question 30

Describe the different tumour types.

Answer 30

1. Benign tumours
   a. May arise in any tissue
   b. Grow locally, do NOT spread to distant sites
   c. Cause damage by local pressure or obstruction
   d. May cause disease
2. In-situ tumours
   a. Usually develop in the epithelium and are usually small
   b. They do NOT invade the basement membrane and supporting mesenchyme
3. Cancers
   a. Fully developed – malignant – tumours that have the capacity to invade and destroy the underlying mesenchyme
   b. Usually highly vascularized (to support tumour growth)
   c. In large tumours, the center tends to be necrotic