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Canc Biology > L2 oncogenes > Flashcards

L2 oncogenes Flashcards

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1
Q

what is an oncogene?

A

A cancer inducing gene which can transform cells.

Usually caused from a transition of a proto-oncogene to an oncogene by a single mutation

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2
Q

when and what was the first tumour virus to be discovered?

A

1910 - Rous Sarcoma virus (RSV)

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3
Q

How was it concluded that in chicken breast sarcomas there must be a transferrable agent? (RSV)

A
  • Chicken breat sarcoma tissue/,muscle was broken up and homogenised with sand
  • filtrate was collected
  • filtrate injected into young chickens - got sarcoma
    (shows must be a transmissible agent in the filtrate (RSV-virus)
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4
Q

what did Temin and Rubin show about RSV?

A

They showed that the RSV persisted in in cell culture when infected - cells showed similar traits to cancerous cells

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5
Q

what effects do tumour viruses have on cells?

A

1) altered morphology - increased thickness of cell layer, loss of contact inhibition, rounded morphology
2) anchorage independent growth - cells form foci

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6
Q

What is meant by anchorage independent growth?

A
  • cells do not require the need to grow/divide on a flattened surface - can grow on top of each other and form foci (large clumps of growing cells)
  • anchor independence correlates with tumorigenecity
  • TGF-B can induce anchorage independent growth
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7
Q

what happens when foci are transplanted into SCID mice?

A
  • predicts tumorigenecity of cells

- tumour develops

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8
Q

what do SCID mice stand for?

A

severe compromised immunodeficent mice

lack thymus, no hair, accept non-celf cells

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9
Q

what time of virus is RSV and what are its featured?

A

Retrovirus - has an RNA genome

  • has envelope and proteins/caspid
  • reverse transcriptase
  • has an extra gene Src (has 4 genes not 3)
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10
Q

How is RSV different to ALV?

A

RSV has an extra gene Src

both have gag, pol and envelope

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11
Q

How does the RNA virus of RSV persist in cell through successive growth cycles?

A

Reverse transcription of viral RNA genome into host DNA

  • Virus enters into cells and sheds envelope
  • Virus RNA and reverse transcriptase is released int the cell
  • RT causes RNA to become DNA - then DNA double stranded helix forms
  • Integration of DNA copy into the host chromosome - PRO-VIRUS
  • translation of RNA causes production of many viral particles (envelope, capsid proteins, RT)- assembly of new viral particles - the virus proliferates and infects new cells.
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12
Q

how can viruses aquire extra host sequences?

A

when viruses come out of genome - if not spliced properly can pull out parts of host DNA - acquire oncogene?
- viruses can acquire an oncogene e.g. Src

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13
Q

why is the theory that proposed mutagens activate latent pro-viruses already in host DNA INCORRECT?

A
  • proved incorrect as
  • do not get clusters of infectious outbreaks of cancer
  • cannot isolate virus particles from human tumour cells (if this was correct there should be viral particles in all tumours - there wasn’t - this cannot be the root cause of cancer)
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14
Q

who discoered reverse transcriptase and linked this to RSV?

A

Temin and David Baltimore

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15
Q

Give an example of other virsuses which can cause human cancer

A

Epstein Barr virus - nasopharyngeal cancer
Hepatitis C virus - hepato celluar cancer
HIV - non-hodkins lymphoma

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16
Q

What is a method that can be used to detect oncogones?

A

Transfection studies

  • expose C3H10T1/1 cells to 3-methylcholanthrene
  • in prescence of calcium phosphate cells take up foreign DNA
  • extract DNA from chemically transformed fibroblasts and inject into normal mouse fibroblasts-(NIH3T3 cells)
  • focus of morphological transformed cells forms
  • inject morpholigcally transformed cells into mouse
  • tumour forms (cells likely to have taken up oncogene (formed from mutations induced by carcinogens-single mutant allele formed)
17
Q

how much of genome is taken up during transfection and what could be concluded from this?

A

0.1% of genome.
10 -6 is probability of 2 unlinked genes having an effect (unlikely more than one gene is mutated)
- more likely a single gene is responsible for transformation (not 2) - oncogenes?
- single mutant allele formed by carcinogens

18
Q

why are transfection experiments important

A

They show that oncogenes can arise in the genomes of cells through mechanisms which have no connection with viral infection - e.g. carcinogens used not viruses

19
Q

what was the first cellular proto-oncogene found?

A

C-ras

20
Q

How can cellular oncogenes be identified?

A

Through repeated rounds of DNA extraction and transfection

  • Extract DNA from foci
  • Add essential bacterial gene
  • split population of DNA into separate tubes
  • inject DNA from different tubes into cells/fibroblasts and see if from foci
  • extract DNA from cells which form foci
  • repeat rounds of transfection
  • ideally will end up with pure poplaiton of oncogene and bacterial gene
  • remove bacterial gene and identify / sequence oncogene.
21
Q

what is a proto-oncogene?

A

normal gene, which when altered by a mutation becomes an oncogene

22
Q

what happens in southern blotting?

A

southern blotting identifies specific sequences of DNA
- fragments separate and are transferred onto a nitrocellulose membrane via the use of a buffer . assay via hybridisation of radioactive/ labelled probes to identify similar sequences

23
Q

what did wigler do to find sequence similar to h-RAS?

A
  • took viral oncogene probe developed from H-ras and probed transformed mouse fibroblasts (NIH3T3 transfromed cells with DNA from human bladder carcinoma) to find sequence highly similar
  • oncogene in transformed NIH3T3 cells has high stringency to viral oncogene h-ras
24
Q

what does K-ras anneal to?

A

K-ras viral oncogenic probe annelas to oncogene from human colon carcinoma cell line

25
Q

how does oncogenic Ras differ from nromal ras?

A
  • oncogenic Ras has a G-T base change - point mutation at position 12
  • glycine - valine aa change
  • causes proto-oncogene to become an oncogene
26
Q

How is burkitts lyphome caused?

A

By TRANSLOCATION AND AMPLIFICATION

  • chromosomes become swapped when cut
  • myc gene moves from chromosome 8 to chromosome 14, downstream of IgH promoter
  • IgH promoter is strong promoter - on a lot of the time - means there is high expression levels of myc gene
  • misregulated expression of myc
  • causes proliferaiton and cancer phenotype
  • western blot shows high L-myc over time in tumour cells.
27
Q

Why does the mutation at position 12 on ras have proliferative effects?

A

Position 12 (glycine) is in area where there is GTPase catalytic activity.

  • mutation at position 12 changes conformation and negatively affects GAPS
  • this means Ras/GTPase cannot change into an inactive form so stays bound to GTP- active ras drive porilferation
28
Q

what occurs in a 1/3 of glioblastomas?

A

The EGF receptor is decapitated/truncated

  • missing the ECD
  • this means the ICD remains activated even if the ligand/EGF is not present
29
Q

what can you do to determine how similar a prob and sequence are?

A

increase the temperature and salt concentration - weaker binding/ less similar matches come apart at lower temperatures.

30
Q

How similar are H-Ras probe and DNA/oncogene of transformed cells?

A

are highly identical
- viral oncogene identical to a cellular oncogene - same thing?

  • evolutionally virus taken oncogene from organisms???

Canc Biology (15 decks)

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