Does UV cause DNA fragmentation?
Posted 03 February 2009 - 12:31 PM
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.
Posted 03 February 2009 - 03:47 PM
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.
Posted 03 February 2009 - 07:49 PM
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.
Posted 03 February 2009 - 09:55 PM
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?
Posted 04 February 2009 - 02:08 AM
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.
Posted 05 February 2009 - 06:27 AM
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?
Posted 05 February 2009 - 08:42 AM
Posted 07 February 2009 - 08:31 AM
Posted 09 February 2009 - 09:11 AM
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.
Posted 09 February 2009 - 04:46 PM
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.
Posted 10 February 2009 - 01:56 AM
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.
Posted 10 February 2009 - 03:18 PM
Posted 06 May 2009 - 12:44 PM
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)