Detergent and Enzyme Treatment of Apoptotic Cells for the Observation of DNA Fragmentation

The uniform degradation of genomic DNA into oligomers of approximately 180–200 bp, or multiples of that, characterizes internucleosomal cleavage of DNA. Such fragmentation is a biochemical hallmark of apoptosis (6). Agarose gel electrophoresis is the most common way to demonstrate the laddering pattern of apoptotic DNA. Traditional purification protocols for the isolation of DNA from apoptotic cells require RNase and proteinase digestion of samples. DNA is then extracted using potentially hazardous solvents such as phenol and chloroform, followed by precipitation. Additional time is needed to dissolve the precipitated DNA into buffer before the sample can be analyzed by agarose gel electrophoresis for the presence of internucleosomal DNA fragmentation. This procedure can take two days. The quantity and quality of DNA recovered from apoptotic cells varies depending on the manipulation of the sample and the protocol used (5). Shearing of DNA during extraction results in the appearance of smears on agarose electrophoretic gels that can obscure the internucleosomal-sized DNA fragments seen in apoptosis. These ambiguous results could easily be misinterpreted as necrosis based on the DNA degradation patterns observed (1,2). In addition, many figures in journals depicting gel electrophoresis of apoptotic DNA lack the complete spectrum of internucleosomal-sized fragments because of the absence of either large or small oligomers, therefore resulting in loss of data (3,4). We demonstrate that solvent extraction and subsequent precipitation are not required for analysis of DNA fragmentation. Only gentle detergent and enzyme treatment of apoptotic cells at 37°C are required to observe DNA fragmentation by agarose gel electrophoresis. Our method provides a rapid test for detection of the smaller apoptotic oligomers produced in a majority of apoptotic cells. It could also be used to analyze large DNA fragments by pulse gel electrophoresis in which genomic DNA is not completely fragmented. This new procedure uses high-percentage agarose gels to increase resolution for smaller oligomers. This allows us to quickly determine whether a cell undergoes apoptotic internucleosomal DNA fragmentation. With 45 min of DNA preparation and 1 h of electrophoresis, we are able to discern that DNA fragmentation has occurred in various cell lines. Thus, preparation and electrophoresis time is greatly reduced, and the yield and integrity of the DNA sample are not compromised by extraction and precipitation procedures. In addition, reduced cost of the reagents and limited exposure to hazardous solvents make this protocol a much more attractive alternative to conventional DNA extraction methods. For demonstration of this method, we induced apoptosis in L929 and WEHI-164 cells with tumor necrosis factor (TNF) and BW5147 cells with dexamethasone. Treatment of L929 and WEHI-164 cells with 1000 U of murine recombinant TNF (Genentech, San Francisco, CA, USA) per milliliter for 18 h induces apoptosis (1). BW5147, a T-cell lymphoma cell line, also undergoes glucocorticoid-induced apoptosis within several hours of treatment with 10-7 M dexamethasone. Treated and untreated cells were collected in 1.5mL microcentrifuge tubes and washed once with 1 mL of 0.02% EDTA in Hanks’ balanced salt solution (HBSS) without Ca2+ or Mg2+. The cells were pelleted by centrifugation for 5 min at 700× g; then 40 μL of the TE lysis buffer (10 mM Tris-HCl, 1 mM sodium-EDTA, pH 7.5) containing 0.25% Nonidet P-40 (NP40) or Triton X100 were added along with 5 μL of an RNase A solution (20 mg/mL) (Sigma Chemical; St. Louis, MO, USA). The mixture was suspended by gentle vortex mixing and incubated for 20 min at 37°C. Then, 5 μL of a proteinase K solution (20 mg/mL) (Sigma Chemical) were added to the sample and incubated an additional 20 min or until the solution cleared. A 6× loading buffer (5 μL of 0.025% bromophenol blue, 0.025% xylene cyanol FF, 30% glycerol) was added to 25 μL of the sample. The sample was then analyzed by electrophoresis on a 1.8% agarose minigel in TE buffer (40 mM Trisacetate, 1 mM EDTA, pH 8.0) for 1–4 h at 36 V. Ethidium bromide-stained DNA was visualized by transillumination with UV light (300 nm) and photographed (5). For fragmentation analysis, the sample preparation time using conventional organic solvent extraction methods required 2 days, compared to 45 min for the technique described above. The induction of internucleosomal DNA fragmentation in various cell lines used was highly discernible using this technique. Figure 1A shows electrophoresis of DNA isolated from L929 cells untreated and treated with TNF for various times. After 6 h of exposure to TNF, induction of apoptosis in L929 cells is clearly evident by distinct oligosomal DNA fragments ranging from 2.4 kb through the final 200-bp fragment (Figure 1A, lane 4). As determined by trypan blue exclusion, 72% of the L929 cells were still viable after 6 h of TNF treatment. DNA samples extracted from the same number of