Week 1 - Models of Carcinogenesis Flashcards
Name the 5 models used to study carcinogenesis.
Mouse skin model Cell culture and in-vitro transformation Transgenic mice Knock-in/Knock-out Mice Genomic Editing
What are the classifications of the two main constituents of coal tar?
Bonus - If you can name the molecules
Mutagen - 7, 12 DMBA
Irritant - TPA
In the mouse skin model, which of the two main types of molecules need to be applied first before the other and why?
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
Explain Knudson’s Two-Hit Hypothesis
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.
Why are primary cell types difficult to grow in culture?
They require complex conditions (physiological).
Name the similarity between growing primary cell types in culture and malignant cell types in culture.
They are both relatively equally as difficult to grow in culture.
What is the underlying significant genetic difference between immortalised cells compared to normal cells?
They have a highly abnormal cell karyotype.
They have undergone major karyotype changes to be able to adapt to growing in culture.
What is an explant?
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.
What is another word for Passaging?
Sub-culturing
What is 4 steps to carry out Passaging?
- Treat the primary culture with protease (Trypsin)
- Cells detach from plastic
- Wash and place into a new medium
- Cells stick down and continue to grow
What is the need for Passaging?
To be able to study cells for a longer period of time/for many generations.
To create immortalised cell lines - 3T3 cells
Name a type of cell which is relatively easy to grow in culture?
Fibroblasts
Name the 4 characteristics for fibroblast growth in culture.
- Anchorage Dependence
- Serum Dependence
- Contact Inhibition
- Finite Lifespan
What is anchorage dependence?
When cells require a surface to adhere to in order to grow.
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?
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
What is contact inhibition?
BONUS What is done to prevent contact inhibition from occurring?
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
What are 3T3 cells?
Immortalised mouse fibroblasts
How are 3T3 cells made?
Continual passaging of mouse fibroblasts - until they stop dividing - spontaneous resumption of cell division occurs, resulting in an immortal cell line.
What are the rules that must be followed when creating 3T3 cells? (2)
Cells must never reach confluence
Passaging must occur regularly to avoid confluence.
How can 3T3 cells be used to study carcinogenesis?
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).
What animal is used to view the effects of cancer 3T3 cells?
What characteristics does the animal have?
Mouse.
Nude Mouse
Genetic defect: nu/nu
Hairless
No Thymus - Required for lack of T-cell immunity which would attack the cells.
What are the characteristics of cancer 3T3 cells?
Not contact inhibited
Infinite Lifespan
Low serum requirement
Anchorage independent growth
How was the RAS oncogene discovered?
Transfection of an area of DNA suspected to be implicated - DNA sequence narrowed down each time to identify specific (350bp) fragment causing foci.
What are the advantages of 3T3 cells in studying carcinogenesis? (5)
- Easy to grow
- Immortal
- Homogeneous Cell Population
- Easy to transfect DNA
- Transform with single oncogene
What are the disadvantages of 3T3 cells in studying carcinogenesis? (5)
- Not normal cells
- Mouse cells - not human
- Fibroblasts - most cancers are carcinomas
- Real Cancers are tissue containing many types of cells
- Immortal - already have first hit
Name 3 alternative cells that could be used instead of 3T3 cells.
- Primary mouse fibroblasts
- Primary human fibroblasts
- Primary human epithelial cells
What is the difference between creating cancerous primary cells compared to immortalised cells (such as 3T3 cells)?
Primary cell types require 2 hits - e.g. MYC + Ras
Immortalised cells already have first hit.
How are transgenic mice made?
Through the injection of DNA into a fertilised mouse blastocyst.
The DNA will integrate into the genome.
Re-implant the egg.
Give 2 forms of transgenic mice.
Knock-In mice
Knock-Out mice
Why are knock-in mice good for studying carcinogenesis?
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.
What is a ‘knock-out mouse’?
A mouse which has had a tumour suppressor in-activated.
Name 2 ways in which DNA can integrate within a genome.
NHEJ - Non-Homologous End Joining
HR - Homologous Recombination
What is NHEJ and explain the process?
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.
What is HR and explain the process?
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.
Name 2 methods of Gene Editing.
CRISPR-Cas9
Zinc Finger Nucleases
Explain how CRISPR-Cas9 mechanism works in bacterial adaptive immunity.
- When infected by a bacterial virus (bacteriophage), some foreign DNA can be integrated into the CRISPR locus
- This can be transcribed to make a “guide RNA” which will recognise the complementary foreign DNA
- This targets a cas9 tracrRNA complex to the foreign DNA
- Cuts and inactivates the bacteriophage DNA
Explain how CRISPR-Cas9 has been adapted for use in gene editing?
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.
