LECTURE 4: Oncogenes

Question 1

How would you check if a gene is a oncogene?

Answer 1

Introduce genes from a cancer genome into normal cells
screen cells for transformants (find foci)
inject them into mice to see if tumors develop
if tumors develop ==> oncogene
identify human oncogenes!

Question 2

Possible problems with oncogene checking experiment

Answer 2

1. How would you get cancer DNA into normal cells?
2. What counts as a normal cell (to put the cancer into)
3. What cancer cells to use for donor DNA?

Question 3

Getting cancer genes into normal cells

Answer 3

Transfection: introduction of DNA into recipient cells.

• Use calcium phosphate precipitation or liposomes (lipid vesicles).
• Cells will take up small amounts of DNA molecules, some DNA becomes integrated into genome (maybe 0.1% of donor genome).
• Very inefficient but workable if you’re working with large numbers of cells in culture

Question 4

What was the aversion to viral transduction?

Answer 4

All…
A) Vector was too efficient, all cells transduced!
B) Viral enhancer/promoter encoded in the LTR
does not mimic real human oncogene regulation
C) Viral integration plus oncogene may cause viral
transformation differently than human oncogenes
D) Viruses convert proto-oncogenes to oncogenes
in ways that wouldn’t occur in human cancer

Question 5

Choosing “normal cells” to have cancer inserted into

Answer 5

Picking 3T3 cells
Immortilization required for appearance of transformed clones
Mouse embryonic fibroblasts grown in vitro can become immortalized cell lines

Question 6

Types of MEF cells

Answer 6

MEFs subcultured on a specific transfer schedule and at specific densities became immortal
3T3 - Transfer every 3 days at 3 x10 5 (low density)
3T12 - Transfer every 3 days at 12 x10 5 (high density)

Question 7

3T3 vs 3T12 growth

Answer 7

Initial decreased growth rates but then they both stabilise
3T3 cells stabilise faster compared to 3T12
Foci would be visible on 3T12, not 3T3 (loss of contact inhibition)

Question 8

Behavior of 3T3 vs 3T12 at high cell density

Answer 8

At high cell density 3T3 cells arrest but
3T12 cells continue to proliferate

Question 9

What hallmark of cancer cells do
3T12s exhibit compared to
3T3s?

Answer 9

Loss of contact inhibition

Question 10

If 3T3 & 3T12 cells were transplanted into mice what would happen?

Answer 10

3T3 are pre-malignant, immortalized cells but they wouldn’t form tumors
3T12 cells are tumorigenic, will form tumors in syngeneic mice

Question 11

The identification of a transforming gene by transfection
EXPERIMENT

Answer 11

Showed was that u could cause cells (3T3) to be transformed by transfecting them with other DNA

Question 12

The identification of a transforming gene by transfection
EXPERIMENT: Procedure

Answer 12

Chemically transformed mouse fibroblasts
Take DNA
Coprecipitated with calcium phosphate
Formed 3T3 Cells

Foci development
Tumor development

3T3 cells are ‘poised’ to become transformed – maybe one more gene can push them over the edge