Apoptosis and Cell Proliferation, страница 6

During DNA synthesis (DNA replication) these modified nucleotides areincorporated into the genomic DNA. Subsequently, those labeled cells are incubatedwith cell death-inducing agents or effectorcells and the labeled DNA is either fragmented or retained in the cell nucleus.Finally each type of DNA (HMW andLMW) is quantitated. Because the labelingof the cellular DNA has to be done prior tothe induction of cell death, this labeling isalso called “prelabeling”.The prelabeling of one cell population (e.g.,the target cells) allows the behavior of thelabeled cells to be traced specifically whendifferent cell populations are mixed.Note: Because cell-mediated cytotoxicity(CMT) proceeds, at least in part, by apoptotic mechanisms, the DNA fragmentationassay may also be used as a CMT assay.In a study of cell-mediated cytotoxicity thetarget cell population is labeled before theeffector cells (e.g., CTL) are added.

Subsequently, due to pore formation in the targetcell plasma membrane, the fragmentedLMW DNA is released from the cytoplasmof the target cell into the culture supernatant (Table 2). The cytotoxic potential ofthe effector cells is measured by quantification of the label released from the damagedtarget cells.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure DNA fragmentationG Figure 4: The biochemistry of DNAfragmentation and the appearance ofthe “DNA ladder”.Because this metabolic prelabeling of thegenomic DNA requires DNA synthesis,only cells proliferating in vitro (e.g., celllines) may be labeled in this way; cellswhich do not proliferate in vitro (e.g., primary cell cultures, tumor cells ex vivo) donot replicate their DNA and therefore, donot incorporate labeled nucleotides (seealso Section 1.3.2.1. “Cellular DNA Fragmentation ELISA” page 54).To detect fragmented DNA in cells whichdo not replicate in vitro, the DNA has to beisolated and analyzed by agarose gel electrophoresis (“DNA ladder assay”, Figure6, see also Figure 4).

Roche MolecularBiochemicals offers a kit, the ApoptoticDNA Ladder Kit, that simplifies this assay.9Cell Death – Apoptosis and Necrosis1Apoptosis Assay MethodsAssays that measure DNA fragmentationnormal cellapoptotic cellHMW-DNAcompartimentHMWLMWNucleus+–Cytoplasm––LMW-DNAHMW-DNA(condensed)HMWLMW–G Figure 5: Compartmentalization of HMW and LMW DNA in normal and apoptotic cells.( = decreasing, = increasing)ApoptosisCell mediatedcytotoxicityCompartmentHMWDNALMWDNAHMWDNALMWDNANucleus++++Cytoplasm–+–+Supernatant–––+G Table 2: Distribution of HMW and LMW DNA in cellsundergoing apoptosis and target cells during cell mediated cytotoxicity.Note: In the early phases of apoptosis, noDNA is released into the supernatant (prelytic DNA fragmentation).

However, in vitro, the apoptotic cells will lyse (“secondarynecrosis“). Therefore, LMW DNA is foundin the supernatant late in apoptosis.10An alternative method which circumventsthe isolation and electrophoretic analysisof DNA is the immunological detectionof LMW DNA (histone-complexed DNAfragments) by an immunoassay (Cell DeathDetection ELISAPLUS, see page 13).This non-radioactive immunoassay, offered by Roche Molecular Biochemicalscan quantitate that hallmark of apoptosis.The Cell Death Detection ELISAPLUS hasbeen designed to quantify DNA fragmentation in cells which do not proliferate invitro (since the kit requires no prelabelingof the cells).

This kit measures the enrichment of histone-complexed DNA fragments (mono- and oligonucleosomes) inthe cytoplasm of apoptotic cells.Each of the methods to detect and measureapoptosis has its advantages and limitations. Because the cellular mechanisms thatresult in apoptosis are complex, most published methods cannot by themselves detect apoptosis unambiguously.To ensure that the mode of cell death in theindividual cell system or experiment isapoptotic, one also has to consider othercriteria like the cellular morphology. Morphologic criteria for apoptotic cell deathinclude, for example, chromatin condensation with aggregation along the nuclear envelope and plasma membrane blebbing followed by separation into small, apoptotic bodies.

When internucleosomal DNAfragmentation is accompanied by thesemorphological features it provides an additional useful criterion to define cell death asapoptotic.Apoptotic DNA Ladder KitCat. No. 1 835 24620 testsTypeDNA purification kitUseful forPreparation of apoptotic DNA fragments for display on electrophoretic gelsSamplesWhole blood or cells in cultureMethodCell lysis, followed by binding of cellular DNA on glass fiber, removal of impurities, and DNA recoveryTimeDNA preparation: < 20 min (after induction of apoptosis)Significance of kit: This kit offers the easiest way to isolate apoptotic DNA fragments for DNA ladder analysis.

The purification method outlined in the kit is muchfaster than other DNA purification methods (e.g., phenol/chloroform extraction,DNA precipitation). Purified DNA maybe mixed directly with gel loading bufferand analyzed on an agarose gel.Test principle: Apoptotic DNA bindsquickly to glass fiber fleece in the presenceof a chaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities are washed off the fleece, the DNAis released from the fleece with a low saltbuffer. The procedure (see Flow Chart 1)involves:1Incubating an aliquot of apoptotic cellswith an equal volume of binding/lysisbuffer. After the incubation, the lysedsample is poured into a filter tube containing glass fiber fleece.1Sample PreparationTreat sample with apoptosis-inducing agent (1–24 h)Incubate treated sample with binding/lysis buffer(10 min, RT°C)Mix isopropanol with sample and pipette mixture intofilter tubeCentrifuge tube assembly (8000 rpm) and discard theflow-through (1 min, RT)Add wash buffer to the filter tube, then centrifuge asbefore (1 min, RT)Repeat the wash step, then add a final high speed spin(13,000 rpm) (1 min, then 10 sec, RT)Insert the filter tube into a 1.5 ml centrifuge tube,and add warm elution buffer to the filter tubeCollect the eluted DNA by centrifugation (1 min, RT)2Using centrifugation to separate theDNA in the lysate (which binds to theglass fiber fleece) from unbound lysatecomponents (which flow through thefleece into a collection tube).3Washing the bound DNA twice.4Eluting the purified DNA from the filter tube and collecting it by centrifugation.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure DNA fragmentationDNA Ladder AssayMix the eluted DNA sample with gel loading bufferApply sample to a 1% agarose gel which containsethidium bromideRun the gel in TBE (Tris-borate EDTA) buffer at 75 V(1.5 h, RT)Place the gel on a UV light box to visualize the DNAladder patternG Flow Chart 1: Assay procedure, Apoptotic DNALadder Kit.11Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure DNA fragmentationSample size: 200–300 µl whole blood orcell suspension (for instance, 2 x 106 cells).The kit allows simultaneous processing ofmultiple samples.Yield1SampleSamplevolumeYield ofpurified DNAWhole blood(human)200 µl3–6 µgCultured cells(K562)2 x 106 cells10 µgKit contents1.

Nucleic acid binding/lysis buffer,ready-to-use2. Washing buffer (ethanol to be addedbefore use)3. Elution buffer, ready-to-use4. Glass fiber filter tubes, 700 µl capacity5. Polypropylene collection tubes, 2 ml(for washes)6. Positive control, apoptotic U937 cells,lyophilizedTypical results: See Figure 6.Specificity: Only nucleic acid will bind tothe glass fiber filters under the conditionsoutlined in the kit. Salts, proteins, andother cellular components do not bind.M+––+–+G Figure 6: DNA ladder assayed with the Apoptotic DNA Ladder KitLane Identification:M = Size marker– = Control cells without camptothecin+ = Cells treated with camptothecinC = Positive control from the kit12–+–+CCell Death Detection ELISAPLUSCat. No.

1 774 425Cat. No. 1 920 68596 tests10 x 96 testsTypeOne-step sandwich ELISA, colorimetricUseful forQuantitation of apoptosis without cell labeling;differentiating apoptosis from necrosisSamplesCell lysates, cell culture supernatants, serum, or plasmaMethodCell lysis, followed by immunochemical determination of histone-complexedDNA fragments in a microtiter plate well (Note: For detection of necrosis,histone-complexed DNA fragments are detected directly in the culture supernatant, without cell lysis)TimeApprox. 3 h (after induction of apoptosis)Significance of kit: This kit quantifies histone-complexed DNA fragments (monoand oligonucleosomes) out of the cytoplasm of cells after the induction of apoptosis or when released from necrotic cells.Since the assay does not require prelabelingof cells, it can detect internucleosomal degradation of genomic DNA during apoptosis even in cells that do not proliferate invitro (for example, freshly isolated tumorcells).

The antibodies used in the assay arenot species-specific, so the kit may be usedto assay cells from a wide variety of species(see “Other applications” in this article).2Resuspending and incubating cells in lysis buffer. After lysis, intact nuclei arepelleted by centrifugation.3Transferring an aliquot of the supernatant to a streptavidin-coated well of amicrotiter plate.4Binding nucleosomes in the supernatantwith two monoclonal antibodies, antihistone (biotin-labeled) and anti-DNA(peroxidase-conjugated). Antibody-nucleosome complexes are bound to themicrotiter plate by the streptavidin.Test principle: The assay uses an one-stepsandwich immunoassay to detect nucleosomes.

The procedure (Figure 7 and FlowChart 2) involves:5Washing the immobilized antibody-histone complexes three times to removecell components that are not immunoreactive.Incubating cells in a microtiter platewell (for instance, 104 human cells in200 µl culture) with an agent that induces cell death (for example, campothecin). After the incubation, the cellsare pelleted by centrifugation and thesupernatant is (containing DNA fromnecrotic cells that leaked through themembrane during incubation) discarded.6Incubating sample with peroxidase substrate (ABTS®).7Determining the amount of coloredproduct (and thus, of immobilized antibody-histone complexes) spectrophotometrically.1Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure DNA fragmentation113Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure DNA fragmentationAAfter incubating cells with an apoptosis-inducing agent,pellet the cells by centrifugation.Discard the supernatant, which may contain necroticDNA that leaked through the membrane during theincubation.Treat cells with apoptosis-inducing agent in the well ofa microtiter plate (MTP) (1–24 h, 37°C)Centrifuge MTP (200 x g ) and remove supernatant(10 min, RT)Incubate treated cells with lysis buffer (30 min, RT)Incubate cells with lysis buffer.Repeat MTP centrifugation (200 x g ) (10 min, RT)1Transfer aliquot of supernatant (lysate) tostreptavidin-coated MTPPellet the intact nuclei by centrifugation.