.=/ 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 19 5243
A simple and rapid method for detection of apoptosis in human cells Frank Rosl Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69 Heidelberg, Germany Submitted July 23, 1992 Programmed cell death or apoptosis is an important regulatory elimination pathway in the development of many animal cell lineages. Apoptosis phenomena have been described in the selective removal of T-cells during clonal selection in the thymus (for review, see ref. 1), during normal erythropoesis (2) and tumour regression (3). Microscopically, apoptosis is characterised by a nuclear collapse which is accompanied by a fragmentation of the cellular chromatin to single or multiple mononucleosomal units mediated by an endogenous endonuclease (4). Extensive apoptosis can easily be visualised by electrophoresis in agarose gels after staining with ethidium bromide (5), whereas the measurement of low levels of nucleosomal autolysis is often accompanied by time consuming prelabeling experiments of total DNA using 3H-thymidine (6) or by hybridisation with 32plabeled bulk cellular DNA (3), sometimes creating high background signals in autoradiography. To circumvent these problems, we devised a simple and quick method to detect apoptosis in human cells. The protocol is based on the assumption that the endonuclease involved in programmed cell death might share some of the proposed biochemical cleavage properties as described for DNAse 1 (7), namely first binding at the minor groove and cutting one strand of the DNA. The mechanism, which is subsequently repeated for the other DNA strand, should create sticky-end like fragments. If such fragmentation products even leave free 3'-OH ends, the resulting nucleosomal ladder should be detected by simply end-labeling the purified DNA by Klenow polymerase. To avoid a nick-translation effect due to the 3' -5' exonuclease activity of the polymerase on randomly nicked or sheared DNA which is not the product of apoptosis, unlabeled nucleotides were omitted and only one 32P-labeled nucleotide was applied for the reaction. The protocol starts with 0.5-1.0 jg cellular DNA, which is treated with 5 U of Klenow polymerase using 0.5 ACi of 32plabeled dCTP (or 32P-dATP) in the presence of 10 mM Tris/HCL pH 7.5, 5 mM MgCl2. The reaction is incubated for 10 min at room temperature and terminated after addition of 10 mM EDTA. The unincorporated nucleotides were removed by three consecutive precipitation cycles of ammonium acetate (2.5 M final concentration)/isopropanol and the labeled DNA was resuspended in 10 mM Tris/HCl pH 7.5, 1 mM EDTA. Between 3000-5000 Cerencov counts were applied on a 1.8% agarose gel, and the probes were electrophoresed for 2 hours at 100 V. After drying the gel on 3 MM Whatman paper, the filter is exposed for autoradiography. As shown in Figure 1, (panel A), apoptotic DNA from tumorigenic cervical carcinoma cells, grown in a nude mouse,
is only barely detectable in an agarose gel (lane b), but can clearly be visualised using the Klenow-labeling procedure (panel B). The same nucleosomal fragmentation pattem can be observed, independently whether 32P-dCTP (lane b) or 32P-dATP (lane c) is used for the fill-in reaction. As a control, DNA from the corresponding cell line grown under in vitro conditions did not show any apoptosis (panel B, lanes d and e). In contrast, DNA from in vitro cultivated primary human keratinocytes, reveals a remarkably level of apoptosis (panel D, lane b), suggesting that this mechanism might also play an important role in cellular senescence. In conclusion, the present protocol provides a simple and rapid method to measure apoptosis from in vivo and in vitro grown cells and should also be helpful in screening experiments for chemotherapeutical drugs, since results can be obtained within one day.
ACKNOWLEDGEMENT I thank Professor Harald zur Hausen for his support and critical reading the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. hi
Williams,G.T. (1991) Cell 65, 1097-1098. Koury,M.J. and Bondurant,M.C. (1990) Science 248, 378-381. Shaw,P., et al. (1992) Proc. Natl. Acad. Sci. USA 89, 4495-4499. Wyllie,A.H. (1987) J. Pathol. 153, 313-316. McConkey,D.J., et al. (1988) Science 242, 256-259. Bertrand,R., et al. (1991) Cancer Res. 51, 6280-6285. Drew,H.R. (1984) J. Mol. Biol. 176, 535-557. a b
a:.
c d
B
a
b
c
d
4m .* VI
.:
D
e
C a b *
I
a
b
.....
i__
1
_
Figure 1. Panel A: DNA from cervical carcinoma cells grown in vivo (lane b) and under in vitro conditions (lane c). Lanes a and d: 123 bp ladder. Panel B: comparison of DNA extracted from a tumor (lanes b and c) with DNA obtained from the same cells grown in tissue culture (lanes d and e) after labeling with 32P-dCTP (lanes b and d) or with 32P-dATP (lanes c and e). Panel C: lane a: 123 bp ladder, lane b: DNA extracted from in vitro cultivated primary human keratinocytes. Panel D, lane b: the corresponding DNA labeled with 32P-dCTP. Lane a in panels B and D represents the 123 bp ladder labeled with 32P-dCTP.
Nucleic Acids Research, Vol. 20, No. 19 5243
A simple and rapid method for detection of apoptosis in human cells Frank Rosl Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69 Heidelberg, Germany Submitted July 23, 1992 Programmed cell death or apoptosis is an important regulatory elimination pathway in the development of many animal cell lineages. Apoptosis phenomena have been described in the selective removal of T-cells during clonal selection in the thymus (for review, see ref. 1), during normal erythropoesis (2) and tumour regression (3). Microscopically, apoptosis is characterised by a nuclear collapse which is accompanied by a fragmentation of the cellular chromatin to single or multiple mononucleosomal units mediated by an endogenous endonuclease (4). Extensive apoptosis can easily be visualised by electrophoresis in agarose gels after staining with ethidium bromide (5), whereas the measurement of low levels of nucleosomal autolysis is often accompanied by time consuming prelabeling experiments of total DNA using 3H-thymidine (6) or by hybridisation with 32plabeled bulk cellular DNA (3), sometimes creating high background signals in autoradiography. To circumvent these problems, we devised a simple and quick method to detect apoptosis in human cells. The protocol is based on the assumption that the endonuclease involved in programmed cell death might share some of the proposed biochemical cleavage properties as described for DNAse 1 (7), namely first binding at the minor groove and cutting one strand of the DNA. The mechanism, which is subsequently repeated for the other DNA strand, should create sticky-end like fragments. If such fragmentation products even leave free 3'-OH ends, the resulting nucleosomal ladder should be detected by simply end-labeling the purified DNA by Klenow polymerase. To avoid a nick-translation effect due to the 3' -5' exonuclease activity of the polymerase on randomly nicked or sheared DNA which is not the product of apoptosis, unlabeled nucleotides were omitted and only one 32P-labeled nucleotide was applied for the reaction. The protocol starts with 0.5-1.0 jg cellular DNA, which is treated with 5 U of Klenow polymerase using 0.5 ACi of 32plabeled dCTP (or 32P-dATP) in the presence of 10 mM Tris/HCL pH 7.5, 5 mM MgCl2. The reaction is incubated for 10 min at room temperature and terminated after addition of 10 mM EDTA. The unincorporated nucleotides were removed by three consecutive precipitation cycles of ammonium acetate (2.5 M final concentration)/isopropanol and the labeled DNA was resuspended in 10 mM Tris/HCl pH 7.5, 1 mM EDTA. Between 3000-5000 Cerencov counts were applied on a 1.8% agarose gel, and the probes were electrophoresed for 2 hours at 100 V. After drying the gel on 3 MM Whatman paper, the filter is exposed for autoradiography. As shown in Figure 1, (panel A), apoptotic DNA from tumorigenic cervical carcinoma cells, grown in a nude mouse,
is only barely detectable in an agarose gel (lane b), but can clearly be visualised using the Klenow-labeling procedure (panel B). The same nucleosomal fragmentation pattem can be observed, independently whether 32P-dCTP (lane b) or 32P-dATP (lane c) is used for the fill-in reaction. As a control, DNA from the corresponding cell line grown under in vitro conditions did not show any apoptosis (panel B, lanes d and e). In contrast, DNA from in vitro cultivated primary human keratinocytes, reveals a remarkably level of apoptosis (panel D, lane b), suggesting that this mechanism might also play an important role in cellular senescence. In conclusion, the present protocol provides a simple and rapid method to measure apoptosis from in vivo and in vitro grown cells and should also be helpful in screening experiments for chemotherapeutical drugs, since results can be obtained within one day.
ACKNOWLEDGEMENT I thank Professor Harald zur Hausen for his support and critical reading the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. hi
Williams,G.T. (1991) Cell 65, 1097-1098. Koury,M.J. and Bondurant,M.C. (1990) Science 248, 378-381. Shaw,P., et al. (1992) Proc. Natl. Acad. Sci. USA 89, 4495-4499. Wyllie,A.H. (1987) J. Pathol. 153, 313-316. McConkey,D.J., et al. (1988) Science 242, 256-259. Bertrand,R., et al. (1991) Cancer Res. 51, 6280-6285. Drew,H.R. (1984) J. Mol. Biol. 176, 535-557. a b
a:.
c d
B
a
b
c
d
4m .* VI
.:
D
e
C a b *
I
a
b
.....
i__
1
_
Figure 1. Panel A: DNA from cervical carcinoma cells grown in vivo (lane b) and under in vitro conditions (lane c). Lanes a and d: 123 bp ladder. Panel B: comparison of DNA extracted from a tumor (lanes b and c) with DNA obtained from the same cells grown in tissue culture (lanes d and e) after labeling with 32P-dCTP (lanes b and d) or with 32P-dATP (lanes c and e). Panel C: lane a: 123 bp ladder, lane b: DNA extracted from in vitro cultivated primary human keratinocytes. Panel D, lane b: the corresponding DNA labeled with 32P-dCTP. Lane a in panels B and D represents the 123 bp ladder labeled with 32P-dCTP.
