Lecture 2 Flashcards
First tumour virus discovered
Rous Sarcoma Virus
How was RSV discovered ??
- Chicken with sarcoma in breast muscel
- Remove sarcoma and break up
- Grind with sand
- collect filtrate passed through fine pore filter
- inject filtrate into young chicken
- Observe sarcoma in injected chicken.
Howard Temin & Harry Rubin showed:
- the virus RSV persisted
- carried out a productive lifecycle in cultured cells (live viral progeny)
- cells displayed traits similar to cancerous cells.
Effects of Tumour Viruses on Cells
Altered morphology
Anchorage independent growth:
Altered morphology (
(RSV in Chick embryo fibroblasts):
• Increased thickness of cell layer
• Loss of contact inhibition
• Rounded morphology
Anchorage independent growth:
• In semi solid medium such as methylcellulose / agarose.
• Cells form foci. (lumps of cells)
Severe Compromised Immuno-deficient mouse (SCID)
• Lack Thymus
• Nude (no hair)
• Will accept non-self cells
• Strong predictor of tumourigenic capacity of the cells.
Xenograft model show
Xenograft model show primary cells injected into mice themselves don’t cause tumours unless already infected with RSV
Evidence viruses cause cancer – most not
RSV
RSV is a retrovirus – sequencing of genome
- Virus - 3 genes gag/pol/env
- 4 genes Src – retrovirus that cause cancer
How does a RNA virus persist in the cell through successive cell growth cycles?
- Temin first suggested the idea of reserve transcription of the viral genome to a DNA intermediate in the 1960s.
- Support for the hypothesis grew on the discovery by Temin and David Baltimore of the enzyme Reverse Transcriptase in 1970.
How does RSV work ??
Dna gets transcribed by cellular machinery
Integration into DNA
DNA inserted randomly and disrutpting regulatory genetic processes
- see diagram
Discovery of viruses led to development of what ?/
Onocogenes
Viral theory of cancer
Guinea pig retrovirus – data produced showing retroviral seq imbedded in DNA of cell or tissue and can excise from it and propagate – maybe conceptually nuclei acid insult activate a process DNA that is Latent in genome excised and activated inducing viral production
• Human Immunodeficiency Virus (HIV)
associated with Non-Hodgkin’s Lymphoma and HIV.
• Epstein Barr Virus (EBV)
associated with lymphoma and nasopharyngeal cancers.
mutagenesis
concept
Something happens to a gene and as a result cells are transformed
Mutagenesis experiment showing transformation
Cells in petri dish embedded in agar undertaking anchorage independent growth
Form colonies visually
– contact inhibition stop as soon as they touch other cells
Cells undergoing transformation aren’t affected by contact inhibition – overgrow each other
Bob Weinberg
Transfection technique
Transfection technique - over view
Take transformed fibroblast and expose to methylcholanthrene (mutagenic drug)
Under normal conditions- take up DNA
Transfection technique
- chemically transformed mouse fribroblast
- add DNA
- inject into normal mouse fibroblast
- formation of a focus of morphologically transformed cells
- inject into mouse host
- Tumour - evidence something has happened to DNA
Work out percentage of genome taken up transformed cell
about 30 genes introduced into each cell
Michael Wigler
First cellular oncogene identified is c-Ras
Michael wigler Experiment
Chemically attached gene to DNA from transformed cell – now with attached essential gene
Might have an oncogene – trying to find it in sea of all the others
Take mixture and put back into mouse fibroblast- foci form from oncogene
Tumour- shows its gone in
Then extract DNA back out – why attached gene essential – can distinguish transfected DNA
Introduce into bacteria without essential gene – only bacteria that grow are those that have tagged DNA that contains oncogene sequence
Select for the only one tagged- keep repeating extract DNA and pass back through Bacteria
Fraction of oncogene
What was the first cellular oncogene to be identified ??
RAS
Southern blotting
- use elecrtophoresis
- agarose gel
- Labelled RNA/DNa
- Sealed bag around gel and labeled probe in buffer
- Labelled Probe hybridises complementary DNA / RNA Band visualised bu autoradiography.
- nitrocellulose gel - binds to molecules
- remove nitrocellulose paper with tightly bound nucleic acid.
- If complimentary to any sequence in the nitrocellulose- anneals at right temp get specific binding then wash away
- Radioactive spot correspond to particular RNA – isolated
Transformed NIH3T3 Genomic DNA interrogated
with H-Ras viral oncogene probe
-implied cancer had a gene similar to human or mice but just mutating it makes it so similar to a viral oncogene probe which showed that humans are related to viruses
Transfored NIH3T3
NIH 3T3 cells were transformed with DNA from a human bladder carcinoma. DNA was extracted and hybridised with probes produced using H-Ras gene from murine sarcoma virus
H- Ras genes
Extra gene - along with gag pol env
How does oncogene ras differ from normal ras ?
sequence - extra gene
Single base change convert cellular ras into a oncogene – glycine to valine
Sufficient to form tumorigenesis
Anchorage independent growth definition
A cell that has lost the need for anchorage dependence, which is essential for cell growth, division, and spreading. Supplement. Cells that have become anchorage-independent are said to have transformed or have become neoplastic in nature.
