Apoptosis - DNA Analysis
During apoptosis, calcium- and magnesium-dependent nucleases are activated which degrade DNA. This means that within the DNA there are nicks and fragmentation. We can detect these in three ways - using DNA analysis to look at a SubG1 peak, using strand break labelling (TUNEL) to detect broken DNA or using Hoechst binding to detect DNA conformational changes.
The Sub-G1 method relies on the fact that after DNA fragmentation, there are small fragments of DNA that are able to be eluted following washing in either PBS or a specific phosphate-citrate buffer. This means that after staining with a quantitative DNA-binding dye, cells that have lost DNA will take up less stain and will appear to the left of the G1 peak.
Because the cells are fixed, we can easily look at a time course following apoptosis induction. Here, we are looking at Jurkat cells following camptothecin treatment.
Click on the image to view diagram in full size
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The advantage of this method is that it is very rapid and will detect cumulative apoptosis and is applicable to all cell types. However in order to be seen in the SubG1 area, a cell must have lost enough DNA to appear there; so if cells enter apoptosis from the S or G2/M phase of the cell cycle or if there is an aneuploid population undergoing apoptosis, they may not appear in the SubG1 peak. Also cells that have lost DNA for any other reason, e.g. death by some other form of oncosis, will appear in the SubG1 region; so we have to be careful about how we define the sub-G1 peak. As with all methods that are used to detect apoptosis, the more processing steps involved, the greater the likelihood of losing cells and as apoptotic and necrotic cells are sometimes preferentially lost, the fewer manipulations the better.
The TUNEL method is often used to detect strand breaks within DNA. TUNEL is an
acronym for Terminal deoxynucleotidyl transferase
mediated dUTP Nick
End Labelling.
The enzyme Tdt is used to add dUTPs to the broken ends of the DNA, these can then
be detected by antibodies with fluorochrome labels. The DNA may simultaneously
be stained with propidium iodide, so it is possible to tell from which phase of
the cell cycle the cells are exhibiting strand breaks. Our Laboratory uses a commercially
available kit for this purpose - the ApoBrdU kit from Phoenix
Flow Systems. In the example below, B cells have been treated with ß-galactosidase
binding protein which has caused cells to become apoptotic in early S phase.
The advantages of this method are that assessment of strand breaks may be correlated with cell cycle status; it is also possible to simultaneously phenotype the cells, and again it is applicable to all cell types. The disadvantages are that it is a technically more demanding technique and, certainly compared to the sub-G1 method, more expensive. The final method we use to assess changes of DNA during apoptosis is to use the DNA binding dye Hoechst 33342 which will enter live cells and stain the DNA in a quantitative manner. However, it has also been observed that in apoptosis, the emission wavelength of Hoechst shifts towards the red end of the spectrum. If we measure blue Hoechst emission (around 440nm) and red emission (above 620nm), apoptotic cells show a shift in fluorescence emission towards the red as illustrated below.
This is a very rapid way of assessing apoptosis but requires the use of a UV laser which are only now becoming available on benchtop flow cytometers.
The protocol for PI staining for sub-G1 DNA is available in pdf or html format.
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