Selection of methods

There are no infallible guidelines on how to chose a procedure that is ideal for the study of apoptosis and its manifestations. The first basic rule is to consider the objective of the study.

• If it is necessary to show simply that cells are dying, cell membrane permeability tests are sufficient and very convenient, e.g. trypan blue exclusion or permeability to propidium iodide.

• If it is to be demonstrated that apoptosis is the mode of cell death that has taken place, simple morphology gives an excellent indication of this process, both in vitro and in vivo, though it is more fashionable to include more expensive procedures such as TUNEL or annexin V studies. When flow cytometric equipment is available for determination of sub-Gl/GO DNA content, this has been found to be exceedingly useful in daily use in the author's laboratory. Demonstration of DNA ladders, if it can be done, is very helpful in validating other procedures in the initial investigation of a new system.

• If the objective is to study mechanistic aspects of apoptosis, any of the techniques described in this volume may be appropriate, but in vitro systems and isolated components of apoptotic cascades are particularly important here. Examples are the release of cytochrome c from mitochondria and a demonstration of the cleavage of procaspase-3 (10).

The second basic rule for the selection of methods for the investigation of apoptosis is to decide which of the following is most important for the study: sensitivity, specificity, ability to quantitate, and speed/economy with which the determinations can be made. Sensitivity can be increased by enrichment for fractions containing dead cells, such as the floating cells in adherent cell populations, but, in general, these choices will vary depending on the circumstances of the individual laboratory, and careful review of the following chapters will provide guidelines for most investigators.

One procedure which is not described elsewhere, provides extraordinary specificity and reasonable quantitation in an in vitro setting, although it requires a significant amount of work and set-up for Southern analysis of DNA. The test depends on the established fact that although the mitochondria show functional aberrations early in apoptosis, the mitochondrial DNA remains intact, while in necrosis, mitochondrial DNA is subject to rapid degradation (9,19).

6.1 Procedure for the determination of the increased mitochondrial to nuclear DNA ratio, for detection and quantitation of apoptosis 6.1.1 Principle

Mitochondria and other cellular organelles are preserved in apoptosis, but undergo rapid degradation in necrosis (4,19). Mitochondria contain multiple copies of a 16.5 kb circular DNA genome which encodes several mitochondrial proteins, and mitochondrial ribosomal and transfer RNAs (20, 21). Nuclear DNA (nuDNA) is degraded in all forms of cell death. In contrast, mitochondrial DNA (mtDNA) remains relatively intact in apoptosis, but becomes degraded in necrosis (9, 19). Thus, determination of the integrity of any mitochondrial gene relative to the integrity of a nuclear gene provides an accurate procedure for distinguishing apoptosis form necrosis. In addition, the ratio of the signal intensity of the mitochondrial to the nuclear gene allows quantitation of the extent of apoptosis in the cell population (9).

DNA extracted from cells consists primarily of nuclear DNA (nuDNA) but also contains mitochondrial DNA (mtDNA), and the mixture can be subjected to restriction enzyme digestion and Southern blot analysis. The integrity of the mitochondrial DNA is determined by the ratio of the abundance of a mitochondrial gene representative of mtDNA (e.g. p72, ref. 22), to the abundance of a nuclear gene representative of nuDNA. An example of such a determination is shown in Figure 7, and its quantitation in Table 3.

Protocol 1. Analysis of cell death by comparison of mitochondrial vs. nuclear DNA degradation assay

A. DNA isolation Equipment and reagents

• Beckman J-6 centrifuge or equivalent

• digestion buffer: aqueous solution of 100 mM NaCI, 10 mM Tris-HCI (pH 8.0), 25 mM EDTA (pH 8.0), 0.5% SDS, 0.2 mg/ml proteinase K (PK)

• chloroform/isoamyl alcohol (24:1)

• phenol/chloroform/isoamyl alcohol (25:24:1) (Ameresco)

• TE buffer (pH 8.0): aqueous solution of 10 mM Tris-HCI, 5 mM EDTA

Method

1. Lyse the cells with the digestion buffer.

2. Incubate the lysed cells at 50°C for 12 h.

3. Extract the lysates once with phenol/chloroform/isoamyl alcohol, and twice with chloroform/isoamyl alcohol.

4. Precipitate the DNA by adding ammonium acetate to 2.5 M, mix well, then add 2 volumes of 100% ethanol.

6. Pellet the DNA by centrifugation at 4000 r.p.m. (12000 g) for 30 min at RT.

7. Wash the pellets with 70% ethanol and repeat the centrifugation step.

8. Aspirate the supernatant and air-dry the pellet.

9. Dissolve the pellet in TE buffer.

10. Take an aliquot for spectrophotometric readings at 260 and 280 nm.

11. The DNA extract obtained as described here can be used for part B.

Protocol 1. Continued

B. Restriction enzyme digestion of extracted DNA, and Southern analysis

Equipment and reagents

• mt-gene probe (e.g. p72, 16S ribosomal • FcoRI (Gibco BRL)

• nu-gene probe (e.g. c-myc) (Oncor) • NaCI, 0.2 M

Method

1. Digest 100 jxg of the DNA samples from part A with FcoRI or H/ndlll (3.5 U/|xg DNA) for 8 h 37 °C.

2. Treat the reaction mixture with 2 units of DNase-free RNase for 1 h at 37°C.

3. Extract the samples with organic solvents as described in part A (step 3).

4. Precipitate the digested DNA samples with 0.2 M NaCI and 100% ethanol, wash the pellets with 70% ethanol, aspirate the supernatant, and air-dry the pellet.

5. Dissolve the pellet in TE buffer and quantitate using a spectrophotometer.

6. Electrophorese 10 |xg of the DNA sample as described in Chapter 3, Protocol 2.

7. Depurinate and denature the samples in the gel as described in ref. 23.

8. Transfer the DNA as described by Southern (24) and immobilize the DNA on the membrane by drying and baking at 80°C for 2-5 h.

9. Nick translate the probes as described in ref. 25.

10. Add probes to the hybridization buffer.

11. Wash the membranes in SSC solution.

12. Wrap the membranes in plastic and expose to autoradiographic film at -80°C for variable periods of time.

13. Analyse the film by densitometric image analysis for intensity.

7. Pitfalls

There is no doubt that apoptosis research will remain a vitally important area of biological and medical research for some time to come. This very fact provides some downsides, however. These include the sometimes exaggerated claims of specificity for methods or reagents, the profusion of reports that may be contradictory, and the expectation of some that the quality of the science

1: Overview of apoptosis HL60-G1 K562

Figure 7. A Southern blot of the DNA shown in Figure 2, hybridized first to a mitochondrial DNA probe lp72), then rehybridized to immunoglobulin lambda constant region gene (O. Note that the mitochondrial gene signal increases during doxorublcin-induced apoptosis of HL60 cells, but decreases during doxorubicin-induced necrosis of K562 cells. In contrast, the nuclear gene signal decreased during apoptosis of HL60 cells, but increased slightly during necrosis of K562 cells. The slight increase in the nuclear gene signal is due to the enrichment of the DNA sample with nuclear DNA because of the loss of mitochondrial DNA, in a project or report may be elevated by tlie simple expedient of including an experiment or two on apoptosis, Tt is therefore probably more important than in any other field of science to maintain a highly critical attitude to assertions that a particular experimental manoeuver results in the occurrence of apoptosis.

The usual precaution is to demonstrate features of apoptosis by more than one independent approach. It is also important to prevent artefactual DNA damage that may mimic apoptotic DNA fragmentation. The loading of intact cells to be lysed in the gel is described in Chapter 3, Protocol 1. Overloading of gels with DNA produces smears rather than ladders, and attention to this point is a simple way to improve one's credibility. Another precaution often overlooked is to establish precisely the diploid DNA content of the cells under investigation before determining the sub-Gl values, since tumour cell populations are not infrequently composed of a mixture of diploid and

Table 3. Ratios of mtDNA to nuDNA during apoptosis in leukaemic cell lines

Cell line Treatment Concentration ImMI

p72/c-myc ratio*

Duration EcoRI Hind III

MOLT-4 Control

Doxorubicin Dox

U937 Control

Teniposide ARA-C

10 10

5 10

12 24

12 12

1.00

1.00

NDb ND

"Nuclear DNA and mtDNA levels were compared directly by sequential hybridization of the same membrane with probes for c-myc as a representative nuclear gene, and p72, a probe for mtDNA. Following densitometry of the autoradiographs, the ratios of the relative values of mtDNA/nuclear gene were obtained for untreated cells ('controls') and for each treated group. Control mtDNA/nuDNA ratios were converted to a value of 1.00, and the 'treated' mtDNA/nuDNA ratios were mutliplied by the same conversion factor yielding the values presented, ±S.E.M.

aneuploid cells. Under these circumstances the diploid peak can mimic the apoptotic sub-Gl peak.

The avoidance of these and other pitfalls and shortcuts should accelerate the acquisition of meaningful advances in apoptosis research.

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