Week 1 - Models of Carcinogenesis

Question 1

Name the 5 models used to study carcinogenesis.

Answer 1

Mouse skin model
Cell culture and in-vitro transformation
Transgenic mice
Knock-in/Knock-out Mice
Genomic Editing

Question 2

What are the classifications of the two main constituents of coal tar?

Bonus - If you can name the molecules

Answer 2

Mutagen - 7, 12 DMBA

Irritant - TPA

Question 3

In the mouse skin model, which of the two main types of molecules need to be applied first before the other and why?

Answer 3

Mutagen then irritant - Mutagen = 1st hit and irritant causes inflammation which increases cell divisions increasing the chance/risk of the 2nd hit/subsequent hits to occur

Question 4

Explain Knudson’s Two-Hit Hypothesis

Answer 4

The Knudson hypothesis, also known as the two-hit hypothesis or multiple-hit hypothesis, is the hypothesis that cancer is the result of accumulated mutations to a cell’s DNA.

Question 5

Why are primary cell types difficult to grow in culture?

Answer 5

They require complex conditions (physiological).

Question 6

Name the similarity between growing primary cell types in culture and malignant cell types in culture.

Answer 6

They are both relatively equally as difficult to grow in culture.

Question 7

What is the underlying significant genetic difference between immortalised cells compared to normal cells?

Answer 7

They have a highly abnormal cell karyotype.

They have undergone major karyotype changes to be able to adapt to growing in culture.

Question 8

What is an explant?

Answer 8

Small piece of tissue taken and grown in culture.

Opposite of implant which is to place tissue/or object into the body - this is taking tissue out of the body.

Question 9

What is another word for Passaging?

Answer 9

Sub-culturing

Question 10

What is 4 steps to carry out Passaging?

Answer 10

1. Treat the primary culture with protease (Trypsin)
2. Cells detach from plastic
3. Wash and place into a new medium
4. Cells stick down and continue to grow

Question 11

What is the need for Passaging?

Answer 11

To be able to study cells for a longer period of time/for many generations.

To create immortalised cell lines - 3T3 cells

Question 12

Name a type of cell which is relatively easy to grow in culture?

Answer 12

Fibroblasts

Question 13

Name the 4 characteristics for fibroblast growth in culture.

Answer 13

1. Anchorage Dependence
2. Serum Dependence
3. Contact Inhibition
4. Finite Lifespan

Question 14

What is anchorage dependence?

Answer 14

When cells require a surface to adhere to in order to grow.

Question 15

What is Serum Dependence, and what is the difference in cellular activity when a cell is in the presence of high serum vs. low serum?

Answer 15

Serum dependence refers to the need for cells to be in the presence of protein rich liquid utilised for cellular mechanisms involved in survival and growth.

Serum Definition: an amber-coloured, protein-rich liquid which separates out when blood coagulates.

High Serum - Motility and Replication
Low Serum - Survival

Question 16

What is contact inhibition?

BONUS What is done to prevent contact inhibition from occurring?

Answer 16

When cells get in close proximity, they signal to one another to stop dividing - motility and replication are inhibited. They become quiescent.

Cells grow to CONFLUENCE - form a monolayer.

BONUS Passaging/Sub-culturing

Question 17

What are 3T3 cells?

Answer 17

Immortalised mouse fibroblasts

Question 18

How are 3T3 cells made?

Answer 18

Continual passaging of mouse fibroblasts - until they stop dividing - spontaneous resumption of cell division occurs, resulting in an immortal cell line.

Question 19

What are the rules that must be followed when creating 3T3 cells? (2)

Answer 19

Cells must never reach confluence

Passaging must occur regularly to avoid confluence.

Question 20

How can 3T3 cells be used to study carcinogenesis?

Answer 20

The cells can be transfected with DNA from tumours via liposomes or calcium phosphate precipitate.

Grow to confluence

View foci of cells, and how cells do not form monolayer (e.g. they stack).

Question 21

What animal is used to view the effects of cancer 3T3 cells?

What characteristics does the animal have?

Answer 21

Mouse.

Nude Mouse
Genetic defect: nu/nu
Hairless
No Thymus - Required for lack of T-cell immunity which would attack the cells.

Question 22

What are the characteristics of cancer 3T3 cells?

Answer 22

Not contact inhibited
Infinite Lifespan
Low serum requirement
Anchorage independent growth

Question 23

How was the RAS oncogene discovered?

Answer 23

Transfection of an area of DNA suspected to be implicated - DNA sequence narrowed down each time to identify specific (350bp) fragment causing foci.

Question 24

What are the advantages of 3T3 cells in studying carcinogenesis? (5)

Answer 24

1. Easy to grow
2. Immortal
3. Homogeneous Cell Population
4. Easy to transfect DNA
5. Transform with single oncogene

Question 25

What are the disadvantages of 3T3 cells in studying carcinogenesis? (5)

Answer 25

1. Not normal cells
2. Mouse cells - not human
3. Fibroblasts - most cancers are carcinomas
4. Real Cancers are tissue containing many types of cells
5. Immortal - already have first hit

Question 26

Name 3 alternative cells that could be used instead of 3T3 cells.

Answer 26

1. Primary mouse fibroblasts
2. Primary human fibroblasts
3. Primary human epithelial cells

Question 27

What is the difference between creating cancerous primary cells compared to immortalised cells (such as 3T3 cells)?

Answer 27

Primary cell types require 2 hits - e.g. MYC + Ras

Immortalised cells already have first hit.

Question 28

How are transgenic mice made?

Answer 28

Through the injection of DNA into a fertilised mouse blastocyst.

The DNA will integrate into the genome.

Re-implant the egg.

Question 29

Give 2 forms of transgenic mice.

Answer 29

Knock-In mice

Knock-Out mice

Question 30

Why are knock-in mice good for studying carcinogenesis?

Answer 30

Transfection of a gene such as MYC means that the cell already has the first hit.

This means there is a rapid onset of tumour development - meaning that they can viewed quicker.

Question 31

What is a ‘knock-out mouse’?

Answer 31

A mouse which has had a tumour suppressor in-activated.

Question 32

Name 2 ways in which DNA can integrate within a genome.

Answer 32

NHEJ - Non-Homologous End Joining

HR - Homologous Recombination

Question 33

What is NHEJ and explain the process?

Answer 33

NHEJ - Non-Homologous End Joining

Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA.

NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair.

Question 34

What is HR and explain the process?

Answer 34

HR - Homologous Recombination

Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.

Question 35

Name 2 methods of Gene Editing.

Answer 35

CRISPR-Cas9

Zinc Finger Nucleases

Question 36

Explain how CRISPR-Cas9 mechanism works in bacterial adaptive immunity.

Answer 36

1. When infected by a bacterial virus (bacteriophage), some foreign DNA can be integrated into the CRISPR locus
2. This can be transcribed to make a “guide RNA” which will recognise the complementary foreign DNA
3. This targets a cas9 tracrRNA complex to the foreign DNA
4. Cuts and inactivates the bacteriophage DNA

Question 37

Explain how CRISPR-Cas9 has been adapted for use in gene editing?

Answer 37

The Guide RNA has been made more stable through the inclusion of a loop.

Instead of having a crRNA sequence corresponding viral DNA, the sequence is adapted to cut any gene, by changing the base sequence.