28. DIY tumours Flashcards
what properties about cells from the embryo make them look like cancer cells?
easy to isolate, culture and proliferate well
what did a paper in the 80s try and do with rat embryo fibroblasts?
try to turn normal cells into tumour cells
what do embryonic fibroblasts not do that cancer cells can?
form foci in soft agar or grow out of the monolayer of tissue culture dish
what two classic oncogenes known at the time were transfected into MEF? what two plasmids were used?
ras and myc
>plasmid containing ras
>plasmid containing ras and myc
what happens when ras is transfected along into REF?
ras was not sufficient to form colonies on soft agar
what happened when ras was transfected into REF with myc? and what does this show?
reasonably number of colonies were formed in soft agar
>this shows the two oncogenes are cooperating in some way
the same experiment using ras and myc was done on MEF, what was concluded from this?
a single oncogene had no effect whereas two oncogenes did, they cooperated to give a phenotype
the same experiment using ras and myc was done on human embryo fibroblasts, what was concluded from this?
this did not work as cancer is harder to instigate in these cells
two papers were published in 1999 and 2001, what did each of them manage to achieve?
they both achieved transformation of human adult tissue into cancerous like state tissue
what did the 2001 paper show? and what three distinct elements did they look at?
human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells
>tHERT
>activated Ras
>SV40 T antigen
describe activated Ras
it is locked in the active state as it cannot hydrolyse GTP for GDP and so is constitutively active
in what order did these factors need to be introduced? (2001) and why is this the case?
> hTERT
T antigen
activated Ras
if ras is put in too early it will activated p16 pathway - p53 needs to be inhibited first by T antigen
what two assays did the 2001 paper do? and what did they see? and what did this show?
colony formation assays in soft agar and tumour formation assays in nude mice
>reasonable number of colonies
>reasonable number of tumour formed
there were less colonies and no tumour in the controls
>this showed that all three factors were needed for transformation
what were control cells transfected with in the 2001 paper?
hTERT and large T antigen and not ras - ras was needed for proliferative capacity
what was noticed about the colony formation assay and the tumour formation in the 2001 paper?
> given the 10^4 cells were plated in soft agar, a limited number of colonies formed (~150)
in breast tissue, only about 50% pf cells led to tumour formation - we would have expect tumour formation to be an all or nothing response
what was concluded from the 2001 paper?
these three factors were not sufficient to cause breast cancer and further mutations in the genome were required - often this is myc
describe what happens when you plate 1 million cells and what happens when you take a colony from this soft agar assay and do a second soft agar assay? and what does this suggest?
> about 1 in 10^2 – 10^5 cells will form colonies are formed from the first 1 million cells
about 1 in 10-10^2 cells will form a colony - we would expect all cells to give rise to colonies as they came from a colon
this suggests that not all cells in a colony are the same
what is seen when foci form a focus formation assay are re-plated?
low number of foci in new population when re-plated
half a million tumour cells are injected into nude mice, what happens? and what does this tell us?
mice develop 1 to 3 tumours
tumour formation is a very inefficient process
tumour cells are taken from mice tumours and re-injected into mice, what happens?
not very many tumours are formed from cells derived from tumours in mice
what do the results from repeated colony formation, foci formation and tumourenesis assay imply?
they imply that not all tumour cells are equal >are equal as some have the ability to form soft agar colonies, foci and tumour in mice but the bulk of the cells do not seem to be able to do this
what dye has two fluorescent peaks when taken up by living cells and binding DNA? and what colours are the peaks?
Hoechst 33258
blue and red
what sort of experiment was carried out to identify the presence of side populations? and what was observed?
flow cytometry, using Hoechst 33258 DNA dye, red fluorescence is plotted against blue fluorescence and a 45 degree line is obtained
>these are clusters of high staining cells and low staining cells
what do low staining cells have that high staining cells do not?
an active pump which pumps dye out of cells
what is the relative size of the high and low staining cell populations?
large population of high staining cells and a smaller population of low staining cells
what two types of cells were these flow cytometry experiments carried out on?
> colorectal adenocarcinoma cell line
>bone marrow
what is the low staining population of cells in the BM? and what does this suggest that the low staining cells in cancer are?
HSC
>that they are cancer stem cells
what two cell types have unlimited proliferative potential?
stem cells and cancer tumour initiating cells
stem cells can be totipotent and pluripotent and differentiate into lots of different cell types. what is the case of cancer cells?
differentiation in cancer cells is not fully understood – there is reasonable evidence that these can differentiate.
how is it possible to identify what tissue a cancer arose in? and what does this suggest?
using histology i.e. the cells from the oligodendroglioma look like oligodendrocytes
>this suggest some degree of differentiation
describe some evidence for tumour differentiation
teeth can be seen in an x-ray of a pelvis, cancers that arise in germ cells can differentiate into an cell in the body
why did we not see very efficient second colony formation from a single foci?
most of the bulk tumour cells are non-dividing, only a few cancer initiating cells are present - these can form foci
what two properties do cancer intiating cells have that are the same as stem cells?
> unlimited proliferative potential
>self-renewal - they can give rise to tumour stem cells and bulk tumour cells
what marker was used to try and identify tumour stem cells? what is this and where is it normally found?
CD133
>five pass transmembrane protein which is heavily glycosylated
>normally found in cell surface of stem cells and not differentiated cells
how was an CD133+ enriched population of cells obtained to further experiment on?
>paramagnetic beads coated in anti-CD133 >cell population exposed to beads >cells exposed to magnet >cells attached to beads attracted to magnet >rest of cells are washed away
how were the CD133+ cells from many different primary tumours assayed? and what was seen? and what did this suggest?
> assayed to see how they proliferate
this was done on bulk cells, CD133+ and CD133- cells
CD133- cells do not proliferate and CD133+ cells do
this suggested that there were two populations of cells that correlated with this surface marker
when differentiation is induced in CD133+ cells what is seen? and what do these cells no longer do?
they lose their CD133 cell mark
>these cells no longer proliferate
when a tumour like kidney cancer is untreated it has proliferative ability. name something that can treat this to reduce its proliferative ability and why this is the case?
IL-15 is a differentiation agent for kidney stem cells
throughout the course of this kidney treatment what expression is lost and what is gained?
oct3/4 - a marker of stem cells - is lost
and E-cadherin expression is gained as cells become more dependent on adhering to their environment
what implication to tumour initiating cells have on therapy?
> we often target the bulk tumours and often base how well the treatment is going based on the size of the tumour
if only a small amount of cells are causing proliferation then we should be targeting these instead
the bulk of a tumour is fairly benign but what problems may it cause?
it may cause pressure damage due to its size
which cells are likely to metastasise?
tumour initiating cells
name four ways that cancer stem cells are able to resist therapy
- drug efflux transporters
- anti-apoptotic mechanisms
- increased DNA damage checkpoint activated and DNA repair
- a relatively quiescent phenotype
what can drug efflux transporters do in addition to pumping out dye? and what is seen when they are blocked?
pump out lots of chemotherapeutic agents
>when efflux transporters are blocked chemotherapeutic agents are more potent
in order to resit apoptosis, what might a cancer stem cell have high levels of and how may this be hard to target?
bcl2
>other cells us the same mechanism
describe cancers stem cells DNA repair
they often have elevated levels of DNA repair
>the impaired DNA repair associated with the bulk of the tumour may not apply to tumour initiating cells
>this makes them more resistant to DNA damage
cancer initiating cells may be quiescent, how does this mean that they can resist therapy?
lots of cancer therapies target proliferating cells
what two types of cell populations can cancer arise from and what needs to happen in one of these cases?
stem cell population
non-stem cells - these mutate and gain stem cell like properties
who showed that four factor can transform somatic cells and what were they?
Yamanaka
c-Myc, Kif4, Sox2 and Oct3/4
if mutations accumulate in stem cells, what may this allow them to do?
excape their normal contrainsts and become tumourogeneic
mutations may arise in somatic cells to give them stem cell like properties, give example of what genes these may be
c-Myc, Kif4, Sox2 and Oct3/4
tumour stem cells have not been identified in all tumour why may this be?
> we might not be looking in the right place for them
>they might not exist
