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BMS247 - Stem cells > Oncogenes > Flashcards

Oncogenes Flashcards

1
Q

In 1970s, what was it thought that most tumours were caused by?

What is the reality?

A

VIRUSES

In reality - only a MINORITY of tumours are causes by viruses

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2
Q

What is the basis of the understanding of human cancers?

Why?

A

The study of tumours

  • Viral genomes are very SIMPLE
  • Have FEW genes
    Therefore, if virus causes a phenotype - likely to know which gene is the cause
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3
Q

What happens when you take a fragment of the tumour caused by Rous Sarcoma virus and transplant it into another animal?

What does this show?

A

Another tumour forms

Shows that there must be something in the tumour that can drive the formation of another tumour when injected into another animal

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4
Q

What did Peyton Rous do?

A

Tries to discover the permissible factor of the Rous Sarcoma virus:

1) Removed the sarcoma from the breast and broke it up into small chunks of tissue
2) Ground the sarcoma with sand - forming an homogenate
3) Collected the filtrate through fine-pore filtration
4) Injected the filtrate into young chicken
5) Observed sarcoma in the injected chicken that was the SAME as the original one

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5
Q

What was the conclusions of Peyton Rous and his chicken experiments?

A

1) Carcinogenic agent very small - pass through a filter
- Must be a virus (they are small enough)
2) Virus could cause sarcoma formation when injected into a chicken in a very PREDICTABLE timetable
3) Could use this virus to induce cancer formation at WILL
4) RSV capable of multiplying within the chicken tissues - large amount of virus can be recovered
- Using this - more experiments can be done

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6
Q

What can RSV do to cells in culture?

Example?

A

Can transform them

Chicken embyro fibroblasts infected with RSV - show traits associated with cancer cells

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7
Q

What are the 2 differences between control cells in culture and the RSV?

A

1) FOCI (clusters) appear after infection

2) Infected cells show a similar metabolism to the cells isolated from tumours

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8
Q

What are foci?

A

Rounded cells grow on top of each other

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9
Q

What is cell transformation?

Where can this be accomplished?

A

Conversion of the a normal cell into a cancer cell:

Can be accomplished in the Petri dish - possible to study cancer cells in vitro

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10
Q

What is cancer?

How can it be studied?

A

The disease of malfunctioning cells

Can be studied at the level of a SINGLE CELL

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11
Q

What are the 2 possible hypothesis of how the transformed phenotype of the initially injected cells is transmitted to the descendants?

A

1) Viral particle must be around ALL the time - to infect all the daughter cells when they are formed
2) Virus is only needed to transform the progenitor cells - which TRANSMIT the phenotype to the descendants

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12
Q

How were the 2 possibilities of trasmission of the transformed phenotype tested?

A

Using ts RSV mutants:

  • Partially defective proteins - function at permissive temperatures (37 degrees)
  • Increase temperature to 41 degrees - protein becomes miss folded and is not functional

Show:
- Transformed state in cells at PERMISSIVE temperature (37 degrees)

  • Transformed state is lost when the cells are at NON-PERMISSIVE temperature (41 degrees) - Virus INACTIVE
  • If lower the temperature again - virus active, transformed phenotype
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13
Q

What is a ts mutant?

What does this mean for RSV?

A

Variants of genes that allow NORMAL FUNCTION of organism at low temperatures

BUT altered function at higher temperatures

In RSV:
- Function @ LOW temperature –> cause transformed phenotype

  • Don’t function @ HIGHER temperature –> Loss of transformed phenotype
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14
Q

What does effects of increasing/decreasing the ts in a RSV show?

A

ESSENTIAL that the virus is CONTINUOUSLY present in the cell to MAINTAIN the transformed phenotype

Shows, that the RSV transforms the cells AS they proliferate

Viral transforming gene is required to both INITIATE and MAINTAIN the transformed phenotype

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15
Q

What are 9 properties of transformed cells?

A

1) Altered MORPHOLOGY (rounded shape)
2) LOSS of CONTACT INHIBITION - ability to grow over one another
3) Ability to grow WITHOUT ATTACHMENT to the matrix
4) Ability to proliferate INDEFINITELY (immortalisation)
5) Reduced requirement for mitogenetic GF
6) High saturation density (large no. of cells in the petridish)
7) Inability to halt proliferation in the ABSENCE of GF
8) INCREASED UPTAKE of glucose
9) TUMORIGENICITY

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16
Q

What happens to normal cells when they contact their neighbours?

A

They STOP growing - form a continuous layer

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17
Q

What do normal cells need in order to grow?

A

Attachment to the matrix

Signals (GF/mitogens)

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18
Q

Why do transformed cells have an increased uptake of glucose?

A

To SUSTAIN cell divison

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19
Q

What is tumorgenicity?

A

The ability to forma a tumour if injected into another animal

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20
Q

What are the genes of a RSV?

What do they do?

A

gag, pol, env - encode viral constituents (common to many viruses)

and a single, EXTRA gene

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21
Q

How was is discovered which gene in the RSV genome was responsible for the RSV-induced transmission?

What did this show?

A

Mutated the genome to remove one gene at a time:

When remove either gag, pol or env:

  • Virus are unable to REPLICATE
  • BUT, virus still able to transform cells
  • Shows these genes are essential for gene replication

When remove the 4th gene:

  • Virus can replicate
  • BUT, fail to induce the TRANSFORMATION of cells
  • Shows this 4th gene is essential to transform the cells
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22
Q

What is the 4th gene called in the RSV genome that induces transformation?

A

src

23
Q

What happens to the src gene when the cells are injected with RSV?

A

src gene is TRANSFERRED to the cells as they become transformed

24
Q

Why can you not use a probe to follow what happens with src from the viral genome?

A

Because there is also a src gene present in the normal organism (c-src)

25
Q

How is c-src present in the genome of a normal organism?

A

As a SINGLE copy

26
Q

What is the difference between the v-src and c-src?

A

c-src - present in the NORMAL organism and is a PROTOONCOGENE

v-src - present in the VIRAL organism and is an ONCOGENE

27
Q

What are the vast majority of cancers caused by?

A

NOT by viruses

By:
1) ACTIVATION of ENDOGENOUS retroviruses

2) INDUCTION by MUTAGENS

28
Q

What are mutagens?

A

PHYSICAL or CHEMICAL agents that INDUCE cancer through their ability to mutate GROWTH CONTROLLING GENES

29
Q

How can it be identified in non-viral oncogenes are present in chemically-transformed cells?

What was the result of this?

What were the conclusions?

A

By transfection experiments:
1) Mouse fibroblasts chemically transformed

2) DNA EXTRACTED from these cells
3) TRANSFECTION to normal mouse fibroblasts using calcium phosphate co-precipitation

Results:
- Formed TUMORIGENIC FOCI that resembles virally infected cells (lead to the formation of a tumour if inject into a different mouse)

Conclusions:
- Donor tumour DNA carried one or several genetic elements (able to CONVERT a normal cell –> a tumourgenic one

30
Q

What is transfection?

A

The transfer of genes from one cell –> another

31
Q

What is the control for the transfection experiments of cells with DNA from chemically-transformed cells?

What were the results?

Conclusions?

A

DNA from NORMAL fibroblasts

Results:
- NO transformation

Conclusions:

  • Something SPECIFIC in the chemically-induced cells
  • NOT from the transfection procedure
32
Q

How do we know only 1 gene from a chemically-transformed cell is needed to transform a fibroblast?

A
  • Only ~0.1% of donor DNA becomes established in the genome
  • Very LOW probability that 2 independant genes are always present in the 0.1% of the DNA transfected into the recipient cell
33
Q

Where do the oncogenes detected in transfection come from?

What is this similar to? How?

A

Derived from pre-existing NORMAL cellular genes LACKING oncogenic function

These genes were CONVERTED by the chemical

Similar to:
- Viral oncogenes (normal gene is converted into a cancer causing gene)

34
Q

Can the SAME group of proto-oncogenes be activated by viruses AND mutagens?

How can this be seen?

A

Yes

DNA probes for retroviral associated oncogenes used on chemically transformed cells:

  • SAME genes found
  • SAME family of proto-oncogenes normally found in the cells can be CONVERTED into oncogenes
35
Q

What is the difference between a proto-oncogene and an oncogene?

A

Proto-oncogene:

  • Normal gene that is involved in the process of cell proliferation
  • Which has the capacity to cause cancer if over-expressed

Oncogene:
- Proto-oncogene that has been MUTATED or OVEREXPRESSED so that normal restraints on cell proliferation is lost

  • Can happen by changing the promoter of the cell
  • Cause cancer
36
Q

How can proto-oncogenes be activated (converted into a proto-oncogene)?

A

By GENETIC CHANGES:

  • Gene under the control of a VIRAL promotor behaves in a completely DIFFERENT way to a gene under the control of a CELLULAR promoter
37
Q

How was H-ras detected?

A

By the transfection of the human bladder carcinoma DNA

38
Q

What is seen when the isolated ras ONCOGENE from transformed cells is compared to the ras PROTOONCOGENE from transformed cells?

A

Size is very similar

39
Q

How can the difference between the ras oncogene and proto-oncogene be determines?

Process of this?

A

Using FRAGMENT RECOMBINATION:

1) Hybrid gene where HALF of the gene from NORMAL cells and half from CANCER cells
2) See if the hybrid could TRANSFORM cells or not
3) Every time a fragment of oncogene was found to be able to transform cell - repeat to NARROW DOWN the area responsible for the difference in behaviour

40
Q

What was found to be the difference between ras proto-oncogene and ras oncogene using fragment recombination?

A
  • 350bp portion needed to induce transformation
  • ONE single amino acid change
  • G12V (Glycine at position 12 –> valine)
41
Q

What do a large number of human tumours carry?

A

POINT mutations in one of the 3 ras genes in the genome

Found that the vast majority carried G12V mutation

42
Q

Why type of molecule is ras?

A

A small GTPase

43
Q

What does ras cycle betwee?

A

ACTIVE GTP-bound

INACTIVE GDP-bound

44
Q

What are GEFs?

A

Guanine exchange factors:

  • Promote the dissociation of GDP from ras
  • So that GTP can bind
  • Activate ras
45
Q

What are GAPs?

A

GTPase-activating proteins:

  • Promote GTPase activity
  • GTP –> GDP
  • Inactivate ras
46
Q

What happens in the Ras G12V mutation?

A
  • Unable to hydrolyse GTP –> GDP (GTPase activity is inhibited)
  • CONSTITUTIVELY ACTIVE
  • Leads to the activation of many downstream pathways
  • Increased PROLIFERATION, INVASION and Cell SURVIVAL
47
Q

In 70% of tumours, what is the mutation in?

A

Myc

48
Q

What are the 3 members of the myc family?

A

C-myc
N-myc
L-myc

49
Q

What happens if myc is over expressed in the nucleus?

A

Growth-promoting transcription factors

50
Q

What does the myc oncogene arise from?

A

3 different mechanisms:

1) Expression driven by the normal promoter but the gene is amplified

  • Tumours contain more than 10 copies of the gene in the genome
  • Gene product expressed at very high levels (higher levels of mRNA)

2) Chromosomal translocation

  • Region of the chromosome fused with a region of a second, UNRELATED chromosome
  • When this happens, c-myc becomes under the control of a foregin transcriptional promoter

3) Pro-virus integration/insertional mutagenesis

  • Virus integrates into the genome IMMEDIATELY ADJACENT to the myc oncogene
  • Oncogene becomes controlled by the VIRAL promoter
51
Q

What does pro-virus integration occur a lot in?

A

In a family of leukaemias induced by ALV ‘avian leukosis virus

52
Q

What type of cancer can chromosomal translocation cause?

What is the translocation between? What does this cause

A

Burkitt lymphoma

  • Translocation between chromosome 8 and 14
  • Expression of myc becomes under the control of an IMMUNOGLOBULIN GENE, which is a VERY active promoter in lympoid cells
53
Q

How can structural changes in proteins lead to oncognes?

A
  • Truncated extracellular portion of receptor - results in CONSTITUTIVELY ACTIVE receptor
  • This happens to the EGF receptor (GF receptor) in cancer –> over activated
54
Q

How does increasing the receptor increase signalling?

A

Dimerisation in the ABSENCE of the ligand

BMS247 - Stem cells (15 decks)

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