12 replies to this topic

[Curtis]

Hello,

I sometimes put my HeLa cells under UV in order to induce intracellular apoptosis but i'm not sure if I can cause DNA fragmentation by the same approach.

I don't have any chemicals to induce apoptosis and that is why I use UV. normaly after 45 min I can observe apoptosis but i'm not sure if 45 min is also enough to get fragmented DNA.

Thank you

[rkay447]

UV creates DNA damage by crosslinking thymidine.  It doesn't cause fragmentation itself but the DNA damage caused is what kicks off your apoptosis which leads to fragmentation, a known step induced by the cell itself when undergoing apoptosis.  

I found the following in a paper.  It's a bit more specific than my explanation:
UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane–pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4PPs) and their Dewar valence isomers.

There are quite a few papers where people have used UV to induce DNA damage and hence apoptosis.  Perhaps you can look at their methods and materials to make sure you are inducing enough damage.

[Curtis]

Thanks rkay447,

I know that intracellular pathway starts from DNA damage and activation of p53 and all but the only thing I'm concerned is that I'm not sure if this DNA damage is the same as DNA fragmentation caused by staurosporine, actinomycin D etc.?

perhaps I can give it a try tomorrow by culturing cells on 10cm2 petri dish and when they are confluent i can put them under uv and extract the DNA at different time points.

[rkay447]

Hmm.. interesting thought.  I would think that the DNA fragmentation associated with apoptosis would be the same for all these methods because DNA fragmentation is a secondary event of apoptosis.  A event rather than a cause.  It a late defining step that occurs for all apoptosis.  I would believe that even though all these compounds and your UV-induced DNA damage activate or inhibit different signalling pathways, they all converge to the same result, apoptosis.  Additionally DNA fragmentation is a very late step in apoptosis since it represents a "point of no return" if you will.  Once a cell starts to digest it's own DNA, there is nothing left but death.  So although there are many potential methods, pathways and signals that can trigger apoptosis, the end event including the DNA fragmentation is the same in all cases.  

Now I say all this with the disclaimer of this is just my own thoughts and not neccessarily made with absolute certainty.  I'm curious of what others think.  Any ideas?

[Curtis]

thanks again,

I cultured the cells on petri dish already, I'm waiting for them to become confluent. maybe by the day after tomorrow I will send you the pics.

[stardust]

Hi there!

Can I just ask quickly how you do the UV treatment? Do you have a special lamp or do you just use the UV light from the gel documentation system? How long and at which wavelenght are you treating you cells?

Stardust

[Curtis]

I do it very unprofessionally. I just open the petri dish cover and put it very close to the UV lamp inside the laminar flow. like between 20-30 cm. this way I can get apoptosis within 30 min. I prefer to put it under laminar flow UV because I can turn on the blower too to make sure no dust sits on the cell surface. remember to remove the petri dish cover, because UV can not go through the cover.

[Curtis]

it didn't work.....I put them under UV for 45min.....I saw the blebbing starting after 25min...but on the gel there was no fragmentation....I used Qiagen's DNA extraction kit, but I didn't use RNAse A to inhibit RNA...I thought it wouldn't be necessary as long as the amount of genomic DNa is very high, and now I can see the double bands of RNA on the gel....next time I need to use RNAse otherwise it won't give me good pictures....I will also kill the cells by virus next time, not by UV

[Curtis]

I keep replying to myself....lol

rkay447,

I did another DNA isolation by a qiagen kit but still I can't see the fragmented DNA....I think these isolation kits only give the genomic DNA...perhaps the fragmented DNA is washed away during the multiple washing steps.

I am sure the new samples must have fragmented DNA cause I killed them with virus.

[bob1]

Your induction of apoptosis is very swift, you must be dosing with a massive amount of UV, which will definitely be causing DNA damage.  Are you sure that it is actually apoptosis?  I thought the general  time line for DNA damage induced apoptosis is about 12-24 hours. Have you checked the caspase 3 expression?  You may be best off getting a known light source (we use a stratalinker also used for southerns and northerns) and try a dose response curve to see if you can catch the right time points.

You will probably not be able to see the fragmentation unless you catch the cells at just the right time to get the classic DNA laddering effect.

[Curtis]

Hello Bob1,

nice to see you back here.

yes, I am pretty sure I see apoptosis by UV irradiation. I've checked with other methods.....I still think the kits give me genomic DNA instead of total DNA.

[bob1]

Genomic DNA is where you would expect to see the laddering, besides the ribosomal and mitochondial components are tiny compared to the genomic, so I would be surprised if you could see it in them.

[asdfyrr]

Hi there.

if you take the dna fragmentation as a marker for apoptosis, you might want to keep in mind that UV light leads to double-strand-breakes which then in turn causes the intrinsic apoptotic pathway to kick in (if the damage is not repairable). Its not that the UV itself would induce strandbreaks, but it causes Thymine-dimers which can lead to failures during DNA repair and in the end, repair-enzyme failure leads to DSBs.

nevertheless, if apoptosis is triggered ( be it because of irradiation, chemicals or death-receptor ligands )it would lead to caspas3activity which also fragments the DNA.

if you want to visualize it ,you could try TUNEL-assay, it visualizes free DNA ends via fluorescence.

i used TNFalpha with success (20ng/ul for 6h in HEK293 cells)