Tumour suppressor genes Flashcards
what does the sendai virus do?
Is a fusogenic agent so can fuse cells
what was the experiment done by Rao and Johnson which demonstrated that some cancer alleles are recessive?
- One cancer and one normal mouse fibroblast (NIH3T3) cell
- fuse cells toegther with sandai virus
- inject hybrid cells into mouse and see if tumour develops
- hybrid cells are not tumourigenic/do not cause tumours when injected into the mice
- this shows the cancer phenotype in these cells is recessive
how can retinoblastoma arise?
- arises sporadically or in families
what are the 2 types of retinoblastoma?
- unilateral- usually in 1 eye
- bilateral - usually in 2 eyes and other areas - familial
what is the difference in the cumulative incidence of Rb tumours and time after diagnosis between unilateral and bilateral Rb patients?
- unilateral patients cumulative incidence is slow
- bilateral patients cumulative incidence increases rapidly
what was shown by Knudsons 1 hit 2 hit hypothesis exp?
- Unilateral Rb patients have a 2-hit theoretical curve - 2 allelic mutations required to cause Rb phenotype
- bilateral patients have a 1-hit theoretical curve (linear) - one mutation pre-disposed so only one required to cause Rb
how can we explain the frequency of the loss of the second allele?
- homologous recombination
- heterozygous segregtation - LOSS OF HETEROZYGOSITY
what is homologous recombination?
where nucleotide sequences are exchanged between 2 similar molecules of DNA which can occur during DNA repair to repair harmful double stranded breaks in the DNA.
What is meant by a loss of heterozygosity in Rb?
- the loss of any functional copies of Rb
what technique proves LOH occurs?
Zymography
what is zymography?
A electroporetic technique used where an SDS gel is inpreganted with a substrate
- an enzyme/protease is run on the gel and where is ends up, it is able to degrade the substrate in the gell and make a hole.
- can identify where the protease what and can determine was isoform of a protease this is due to differences in MW.
what protease is commonly used in zymography in Rb patients and why?
Esterase D as it is located on the chromosome close to Rb
(esterase D is always lost in tumour tissue)- like Rb
where does LOH occur in Rb?
On chromosome 13q14
How can you tell if chromosome has undergone heterozygosity looking at esterase D?
- there are 2 isoforms of esterase D (version 1 smaller than version 2)
- if there is only 1 isoform found on the gel - LOH has occurred
What is the LOH in neurofibromatosis and give some features of this
LOH of NFI (neuronal specific GAP) which converts active Ras into inactive Ras.
- get caffe latte on skin and effects nerous tissue
where is LOH in adenomatous polyposis?
- LOH in APC gene (5p21)
- means that GSKB cannot degrade Bcatenin as degradation complex doesn’t form efficiently
- Bcatenin accumulates and enters the nucleus espite absence of Wnt
- causes gut stem cell proliferation to rise
what is used to find tumour supressor genes?
Loss of heterozygosity
what is a newer technique (than zymography) which shows that LOH has occurred?
- restriction fragment length polymorphism (RFLP)
- probe recognises certain sequence on maternal and paternal chromosome
- maternal chromosome is frgamented as cleaved by ecoR1
- when run on gel with probe there is no longer 1 allele of each chormosome
what technique has overtaken from RFLP?
SNP analysis
- PCR based approach
- primers recognise a particular sequence known to vary in a population
- if there is a nuceotide difference, primer fails to bind, no elongation so no product is produced - shows LOH
which chromosomes show lots of LOH in colon cancer?
- lots of LOH on Chr 17 and 18
not much going on on short arm of chr13/14
what is the LOH in von hipple syndrome and give features of this syndrome?
- LOH in VHL
- kidney cancer - familial
Why does a lack of funciton VHL cause cancer?
- VHL recognises and binds to HIFa in normoxic conditions and induces the degradation of HIF1a in the proteosome
- when VHL is mutated it cannot bind and degarde HIF1a
- This means pro-angiogenic genes can be transcribed in normoxic conditions such as VEGF
- angiogenisis occurs even in normoxic conditions - beneficial to cancer cells.
what modifies HIFa in normoxic conditions?
- proline hydrolase
- adds OH groups onto HIF1a
- means HIF1a can be recognised by VHL and then undegoes degradation
what does HIF stand for?
hypoxia inducing factor
what happens to HIF in hypoxic conditions?
proline hydroxylase has no effect in hypoxic conditions
- HIF1a is not modified so cannot be recognised and degraded by VHL
- HIF1a bind to pro-angiogenic genes (e.g. VEGF) with HIF1b which causes transcription of VEGF - angiogensis can occur
Define loss of heterozygosity
Loss of heterozygosity (LOH) is where one of two alleles at a heterozygous locus is lost. Is either discarded or replaced with a second copy of the surviving allele
Give another way in which LOH can occur besides mitotic recombination
Gene conversion
Jumping polymerases
