Cancer

Question 1

What are the different types of carcinoma?

Answer 1

Basal cell carcinoma (skin)
Squamous cell carcinoma
Transitional cell carcinoma (transitional epithelium is found in the bladder)
Adenocarcinoma

Question 2

What are the names given to malignant tumours of striated muscle, smooth muscle and the nerve sheath?

Answer 2

Striated muscle – rhabdomyosarcoma rhabdo= rod shaped
Smooth muscle – leiomyosarcoma leio=smooth
Nerve sheath – Malignant peripheral nerve sheath tumour

Question 3

What is the term given to a MALIGNANT tumours that show little or no differentiation?

Answer 3

anaplastic

Question 4

name some proto oncogenes and what they code for normally

Answer 4

Myc - tyrosine kinase
Jun - tyrosine kinase
Ha Ras - G protein
Ki Ras - g protein

Question 5

name some TSG

Answer 5

BRCA1
p53
APC (mutation causes FAP)

Question 6

what is the name of the checkpoint between metaphase and anaphase

Answer 6

spindle assembly checkpoint- this is a checkpoint where eg BUB proteins fall off the kinetochores when the spindles attach to them. Once all BUBs fall off, the cell procedes to next phase

Question 7

Name four different types of spindle attachment.

Answer 7

Amphitelic – normal spindle attachment
Syntelic – both kinetochores of the sister chromatids are attached to spindles from one centrosome
Merotelic – one kinetochore of one of the sister chromatids is attached to spindles from both centrosomes
Monotelic – one kinetochore of one of the sister chromatids is attached to a spindle, the other is unattached

Question 8

which 3 aa are phosphorylated

Answer 8

serine, threonine and tyrosine , all contain OH groups for phsophorylation

Question 9

How does herceptin work

Answer 9

Herceptin – inhibits the Her2 tyrosine kinase receptor (important in many tumours e.g. breast)

Question 10

name an important adaptor protein and its structure

Answer 10

Grb2
It is modular
It has an SH2 domain, which binds to the docking sites (phosphorylated tyrosine residues on the tyrosine kinase receptors)
It has two SH3 domains are proline rich regions

Question 11

How is RAS turned off

Answer 11

by GAP proteins with trigger the intrinsic GTPase activity within RAS to form RAS-GDP (inactive)

Question 12

2 ways for RAS to be mutated? Hint - V21Ras and L61Ras

Answer 12

V21Ras – glycine is replaced by valine, which means that a simple hydrogen side chain is replaced by a hydrophobic sidechain. This hydrophobic side chain doesn’t allow GAPs to bind to Ras, thus preventing inactivation of Ras.
L61Ras – glutamine is replaced by leucine (in position 61), which means that an amine side chain is replaced by a hydrophobic side chain. This inhibits the GTPase activity of Ras so Ras remains in the active, GTP bound form.

Question 13

3 kinases involved in the RAS pathway

Answer 13

Raf
which activates Mek
which activates ERK
ERK eventually phosphorylate proteins involved in gene regulation (eg transcription factors) which eventually lead to turning on of Myc gene- involved in cell proliferation. See slide 26 for what happens after transcription of these early genes

Question 14

What does the mitosis-promoting factor (MPF, also called maturation promoting factor) consist of? How can it be activated?

Answer 14

CDK1 + cyclin B slide 23
Activating phosphorylation by CAK (Cdk activating kinase)
Removal of the inhibitory phosphorylation (that was placed by Wee1)by Cdc25

Question 15

Which Cdk/cyclin is required for G1/S phase?

Answer 15

Cdk2-cyclin E = G1/S cdk complex

Question 16

Which Cdk/cyclin is required for S phase?

Answer 16

Cdk2-cyclin A = S cdk complex

Question 17

Describe the role of retinoblastoma protein in the quiescent G0 state.

Answer 17

Holds onto E2F (a transcription factor) so that E2F cannot transcribe proteins

Question 18

2 families of CDK inhibitors (CKI)

Answer 18

INK4 family

CIP/KIP family

Question 19

How do CKIs work (by family)

Answer 19

INK4 – G1 phase – it displaces cyclin D from the Cdk4/6-cyclin D complex
CIP/KIP – S phase – it binds to the Cdk/cyclin complexes and inhibits them
NOTE: these inhibitors need to be degraded at various stages for the cell cycle to progress

Question 20

types of DNA mutation

Answer 20

Base dimers and chemical cross-links (chemicals cross link DNA together)
Base hydroxylations
DNA alkylation (used in chemo to target cancer DNA)
Abasic sites
Single strand breaks
Double strand breaks
DNA adducts

Question 21

B[a]P metabolism and how it is carcinogenic

Answer 21

B[a]P is a substrate for CYP450, which converts it to an OXIDE: B[a]P-7,8-oxide (this is an electrophile)
The body has a defence mechanism – epoxide hydrolase converts the oxide to a DIHYDRODIOL (B[a]P-7,8-dihydrodiol)
This is inactive
However, this dihydrodiol is also a substrate for CYP450, which converts it to another OXIDE (B[a]P-7,8-dihydrodiol-9,10-oxide)
This even more reactive than the previous oxide – it goes on to form DNA adducts

oxide, dihydrodiol, more reactive oxide

Question 22

2 bladder carcinogens (dyestuff)

Answer 22

2 napthylamine

benzidene

Question 23

2 napthylamine carcinogenic mechanism

Answer 23

1. Firstly, 2-naphthylamine is converted by CYP450 to a hydroxylamine derivative, which is reactive
2. In the liver, this is glucuronidated (thus inactivating it)
   3.The inactive metabolite is excreted by the liver and then it goes to the bladder where it mixes with the urine
   4.The ACIDITY of the urine causes hydrolysis of the glucuronide groups- this forms a nitrenium ion
   This is electrophilic so it leads to the formation of DNA adducts

Question 24

What type of DNA mutation can UV cause

Answer 24

pyrimidine (esp. thymine) dimers

Question 25

What are the consequences of oxygen free radical attack on DNA? (3)

Answer 25

Ionising radiation promotes free radical formation which have an unpaired electron and so are electrophilic - attacks DNA. This causes
1. DNA single stranded or double stranded breaks
2. Apurinic/apyrimidinic sites (cuts damaged DNA out but fails to replace/refill)
3. Base modifications
ring-opened guanine & adenine
thymine & cytosine glycols
8-OH-adenine & 8-OH-guanine

Question 26

types of base modifications as a result of free radical attack

Answer 26

ring-opened guanine & adenine
thymine & cytosine glycols
8-OH-adenine & 8-OH-guanine

Question 27

4 types of DNA repair

Answer 27

Direct reversal of DNA damage
Base excision repair - remove bases
Nucleotide excision repair - remove only nucleotides
During- and post-replication repair

Question 28

2 examples of direct reversal of DNA damage

Answer 28

Photolyase looks for cyclobutane-pyrimidine dimers and cuts them
Methyltransferases and alkyltransferases remove alkyl groups from the bases

Question 29

Base excision repair

Answer 29

DNA glycosylase hydrolyses between the base and the sugar (this bond is a glycosidic bond hence glycosylase)

Then AP endonuclease splits the backbone so there is a gap in the backbone (endonuclease break phosphodiesterbonds)

DNA polymerase then fills in the missing base (using the complementary strand as template)

DNA ligase then seals the DNA by filling in the backbone

Question 30

Describe the process of nucleotide excision repair.

Answer 30

Endonuclease makes two cuts in the DNA on either side of the site of damage (this demarcates a patch of DNA)
Helicase then removes this patch, leaving the double strand with a patch missing
DNA polymerase replaces the missing bases
DNA ligase joins the DNA up

Question 31

3 Fates of DNA damage

Answer 31

low damage - repair
high damage - apoptosis
any damage with faulty repair = aberrant proteins/ cancer if at critical genes eg TSG or oncogenes

Question 32

how to test for carcinogenic properties

Answer 32

Look at structure of compound
Test in vitro on bacteria (Ames test)
Test in vitro on mammalian cells
Test in vivo on mammals

Question 33

describe the Ames test

Answer 33

This test usually uses Salmonella typhimurium
The bacterium is genetically engineered so that it can’t produce histidine, so it can only survive and grow on a culture medium that has exogenous histidine
The compound to be tested is, firstly, incubated with rat liver enzymes containing CYP450 enzymes to metabolise the chemical into an active form that can be carcinogenic
The bacteria are mixed with the active chemical and then placed on a culture medium with NO histidine
Any colonies that survive will have become mutated by the chemical so that it regains the ability to produce its own histidine and hence cangrow in the absence of histidine
Any bacteria that hasn’t been mutated will die on the dish
The greater the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine

Question 34

How can you test carcinogenic properties on mammalian cells? How can you do this test in vitro and in vivo?

Answer 34

eg micronucleus assay
in vitro give chemicals that cause breakage of DNA and allow cell to divide. Stop cell just before cytokinesis so that it is binucleus and you will seen and extra micronucleus

in vivo do the same thing on bone marrow multipotent cells and look for micronucleus in peripheral erythrocytes (they cannot remove the micronucleus)

Question 35

What are the 2 types of DNA damage that can be detected using the micronucleus assay

Answer 35

Clastogenicity – chromosomal breakage

Aneuploidy – chromosomal loss/change in the number of chromosomes

Question 36

What can cause aneuploidy?

Answer 36

Mis-attachment of the microtubules to the kinetochores

Mitotic checkpoint defect

Aberrant centrosome/DNA duplication (abnormal number of centrosomes leads to abnormal division of the chromatids, and abnormal cytokinesis)

Question 37

taxanes and vinca alkaloids mechanism

Answer 37

These alter microtubule dynamics and produce unattached kinetochores, which leads to mitotic arrest. (if you split the chromsomes unevenly due to the drug, then the daughter cell will not be able to divide further= mitotic arrest)