Cancer Biology 2 Flashcards
Gene alteration that activate proto-oncogenes are sometimes small and cannot be seen through microscopy or staining (as other mutations can). DNA transfection can identify c-oncs that have become activated by point mutations. Describe this process.
Transformation of mouse fibroblasts
This DNA was was introduced into normal mouse fibroblasts by calcium phosphate co-precipitation
Formation of s focus of morphologically transformed cells
Clone the transfected oncogene from the focus or analyse by southern blotting
DNA samples from NIH3T3 foci (in DNA transfection procedure) were analysed by southern blotting . Which mutant genes were identified? Which codons are they often found in?
Ras gene family (K-Ras, H-Ras and N-Ras)
Introduction to normal NIH3T3 cells showed that the mutant ras genes led to formation of transformed foci- Ras mutations were identified in 3 codons- 12, 13 or 61
Ras mutations are found in almost all human cancers
Proto-oncogenes are represented in most biological processes. Name a few processes in which some are found
Growth factor receptors, nuclear transcription factors, protein kinases
How is tyrosine phosphorylation implicated in human cancers?
Receptor tyrosine kinases are a type of tyrosine kinase. What can happen if there is a phorylation cascade in which intracellular proteins are phosphorylated?
Tyrosine kinases (enzymes) mediate signalling in many cells, determining processes such as growth, differentiation, senescence and apoptosis. In normal cells they are tightly regulated, but if the acquire a mutation, they may be over expressed. They can be stuck ‘on’ and so constantly signal for growth
Receptor tyrosine kinases can cause cascades of signalling if mutated, this signal can be transduced to the nucleus by phosphorylating intracellular proteins and this may actively change expression of certain genes (causing growth)
The epidermal growth factor receptor (EGFR) gene was cloned. They found a sequence homologous to the Src gene found in chickens. What does this suggest about the tyrosine phosphorylation activity potential of EGFR?
What is the v-ErbB oncogene? Which two types are found in human cancers?
EGFR may have the potential to be involved in human cancer signalling pathways and its tyrosine kinase activity may be relevant to an oncogene
v-ErbB oncogenes are truncated versions of EGFR that lacks the extracellular domain (that binds EGFR to EGF).
This family contains receptor tyrosine kinases and have homologous regions to a leukaemia viral oncogene. Two important ones are HER1 and HER2 (found in breast cancer)
Cancers are often characterised by switchig from paracrine signalling to autocrine (so auto-stimulates itself- produces signals for itself). The human epidermal growth factor receptor (HER) is complex. Which HER is the classic EGF receptor?
HER 1 but EGF can also bind to HER 2
Growth factors work in different ways to achieve dimerisation of the receptors at cell surface. Describe two.
Some growth factors are themselves dimeric and so bring two receptor molecules together when they bind
Some bind as monomers and induce a conformational change in the receptor chains that lead to their dimerisation
What mechanism of dimerisation of the growth factor receptors is seen in human cancers? In which oncogene is this mechanisms seen?
Point mutations in the transmembrane domain of the receptor, or truncation of the receptor’s extracellular domain, can lead to spontaneous dimerisation and subsequent dowmstream signalling in the absence of growth factor. This truncation is seen in the v-ErbB oncogene
Note- This is associated with gene amplification that leads to over expression of receptors, which then auto-stimulate- this is seen in many human cancers
What is trans-phosphorylation?
When receptor dimerisation leads to activation of its tyrosine kinase activity, resulting in phosphorylation of its cytoplasmic domains (each chain of the receptor is phosphorylated by the kinase domain of the opposite chain)
What three domains are found in Src which are conserved in other signalling molecules too?
SH1, SH2 (recognises phosphotyrosine) and SH3 (recognises polyproline/proline-rich sequences)
What does the arginine residue on the SH2 domain do?
Specifies interaction with phosphotyrosine instead of tyrosine
Src is a tyrosine kinase itself, therefore it is important to keep it inactive until it binds to a phosphotyrosine-containing receptor. How do c-Src and v-Src differ?
c-Src is inactive until binding
v-Src is constitutively active
How is c-Src kept inactive?
Autoinhibited by intramolecular interactions
These molecules then enhance substrate binding when the oncogene is to be activated and this allows phosphorylates tyrosine residues on many substrates to be implicated in many areas of the cell
What does Grb2 act as for the phosphorylation of the EGF receptor? What happens after/what does this induce?
Links SOS to the EGFR
SOS then interacts with Ras molecules
This induces release of GDP from Ras to bind with GTP
This leads to conformational change which exposes Ras ‘effector loop’
Interacts with several downstream signalling partners that are known as Ras effectors- this leads to further effects on the cell
Summary: what does inappropriate activation lead to in cancer cells?
Pleiotropic effects
