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Nucleic Acids Research Pages 4422-4425  

Homogeneous rate of degradation of nuclear DNA during apoptosis
Introduction
Materials And Methods
   Cell line
   UV irradiation and cell culture
   DNA isolation and Southern blots
   DNA probes and quantification
Results
   UV irradiation induces HPCG17 cells to undergo apoptosis
   DNA fragmentation occurs at all stages of cell cycle
   DNA degrades at same rate regardless of transcriptional state or location
Discussion
References

Homogeneous rate of degradation of nuclear DNA during apoptosis

Homogeneous rate of degradation of nuclear DNA during apoptosis

David B. Winter, Patricia J. Gearhart and Vilhelm A. Bohr

Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA

Received June 12, 1998; Revised August 11, 1998; Accepted August 18, 1998

ABSTRACT

DNA fragmentation during apoptosis is characterized by endonucleolytic cleavage of chromosomal DNA into an oligonucleosomal ladder. To determine if actively transcribed genes are more susceptible to cleavage during apoptosis than non-transcribed genes, the rate of fragmentation of differentially expressed genes was measured in B-lymphocyte hybridoma cells. Five genes were studied based on their transcriptional activity and/or nuclear localization, and mitochondrial DNA was assayed as a negative control for apoptotic fragmentation. Apoptosis was induced in the hybridoma cells by ultraviolet light, and DNA was prepared at multiple time points after ultraviolet irradiation. Degradation into an oligonucleosomal ladder appeared as early as 2 h after treatment, showing that fragmentation is rapidly activated in hybridoma cells. The DNA was then digested with restriction enzymes, separated by gel electrophoresis and hybridized with the gene-specific probes for Southern blot analyses. Loss of gene-specific signals was measured by quantitation of autoradiographs. The results show all of the nuclear genes were degraded at the same rate regardless of their transcriptional status or nuclear localization. The data suggest that once the cell activates its destruction program, nuclear DNA is rapidly degraded in a homogeneous manner.

INTRODUCTION

Apoptosis is a genetically programmed formula for death that is utilized in pathways of development, building of tissues and tolerance of lymphocytes that react to self-proteins (1-4). One of the distinctive features that differentiates apoptotic death from necrotic death is the pattern of DNA fragmentation. Nuclear, but not mitochondrial, DNA in apoptotic cells undergoes a unique, stepwise pattern of fragmentation over time (5-9). During the first step, degradation starts as early as 5 min after the onset of apoptosis and requires Mg2+ ions. Chromosomal DNA is cleaved into large 600-750 kb fragments and then into smaller 50-300 kb fragments (7,10-12). During the second step, further degradation continues over 2-24 h and requires both Mg2+ and Ca2+ ions (7,13). The fragments are cut into much smaller pieces to form an oligonucleosomal ladder of ~200 bp increments. Each subsequent step in fragmentation depends upon the step before it, and the process may be halted by the removal of the appropriate cations (7,14,15).

Concurrent with the fragmentation of DNA, there are other changes in the nucleus that can be observed by histology. The euchromatin and heterochromatin begin to condense and aggregate around the nuclear envelope while the nucleolus stays intact (16). This uneven organization of chromatin may lead to differences in accessibility of certain regions of DNA to the endonucleases that are activated during apoptosis. The accessibility of DNA has been shown to influence several DNA-associated processes. For instance, there are many examples in the literature showing that transcriptional state of a gene determines whether its sequences will be readily available to modification by enzymes or chemical agents. In immunoglobulin genes, rearrangement of variable gene segments (17), heavy chain class switching of constant genes (18) and somatic mutation of variable genes (19) are all linked to their transcriptional status. In DNA repair, transcribed genes are repaired faster than non-transcribed genes (20-22). The rate of repair depends, at least in part, on the location of a particular gene in the euchromatin or the heterochromatin, and on its accessibility to the repair machinery (23,24).

To determine if DNA accessibility plays a role in determining the rate of degradation in genes after the induction of apoptosis, we measured the relative rates of fragmentation of five nuclear genes in a hybridoma cell line that was exposed to ultraviolet (UV) light. The genes were selected based on their transcriptional state in B-cell hybridomas and their location in the nucleus. For location, we assayed the ribosomal sequences that are located in the nucleolar region where the chromosomal packaging and other aspects of DNA function are unique (16). We also measured the rate of degradation of mitochondrial DNA during apoptosis, which has been shown to not undergo the same fragmentation observed in nuclear DNA (6). The results show that degradation is not affected by either the transcriptional state of the nuclear genes or their location in the chromatin.

MATERIALS AND METHODS

Cell line

The HPCG17 cell line is a murine hybridoma made from BALB/c B cells that secretes antibody specific for the phosphorylcholine antigen (25). Cells were grown in suspension in RPMI#1640 with 10% fetal bovine serum.

UV irradiation and cell culture

Approximately 5 × 108 cells were exposed to UV irradiation as previously described (26). Briefly, the cells were grown to a density of 0.5 × 106 cells/ml (>90% viable), chilled to 4°C, washed with cold phosphate-buffered saline, transferred into Petri dishes at12.5 × 106 cells/ml and exposed to 40 J/m2 of UV light. The hybridoma cells were then resuspended in media to a density of0.5 × 106 cells/ml and placed in an incubator for up to 24 h. Aliquots were removed at 2 h intervals and incubated with 0.2% Trypan blue. Viability was assessed by uptake of the dye and appearance of blebs in the membrane (27). The cell cycle profile of cells undergoing apoptosis was determined by staining ethanol-fixed cells with propidium iodide (Boehringer Mannheim, Indianapolis, IN) and measuring DNA content of individual cells with a flow cytometer.


Figure 1. Rate of cell death in HPCG17 hybridoma cells following UV irradiation. Cells received 40 J/m2; viability was assessed by Trypan blue exclusion and formation of membrane blebs.

DNA isolation and Southern blots

Approximately 80 × 106 cells were removed every 2 h and DNA was isolated by proteinase K digestion, phenol/chloroform extraction and RNase digestion. Sixty µg of DNA was cut with BamHI, run in triplicate (10 µg/well) on a 0.8% agarose gel under alkaline conditions for 16 h and transferred to a Sureblot[trade] nylon membrane (Oncor, Rockville, MD). The membrane was pretreated with Hybrisol II[trade] (Oncor, Rockville, MD) for 4 h before each probing and stripped with 1 M NaOH between probings.

DNA probes and quantification

The following murine probes were labeled with [[alpha]-32P]ATP by the random priming method (Ready to Go random prime kit, Pharmacia Biotech, Piscataway, NJ): a 6.7 kb probe detecting the 28s ribosomal gene (28), a 1.9 kb fragment specific for the [beta]-actin gene (29), a 1 kb probe for the intron between the immunoglobulin [kappa] joining and constant gene (Ig[kappa]) (30), a 150 bp probe for the CCAAT/enhancer-binding protein (C/EBP[alpha]) (31) and a 520 bp fragment specific for type II collagen (32). A 2.5 kb probe for the mitochondrial genome (33) was labeled with [[alpha]-32P]ATP using a SP6/T7 transcription kit (Boehringer Mannheim, Indianapolis, IN). The blots were hybridized for 12 h and washed at 60°C for 30 min in 0.2 × SSPE and 0.1% SDS. Gene-specific bands were detected and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The percentage of non-degraded DNA at each time point was determined by dividing the PhosphorImager value for the amount of DNA at that time by the value for the amount of DNA present at 0 h and then averaging the triplicate determinations.

RESULTS

UV irradiation induces HPCG17 cells to undergo apoptosis

Both normal and transformed lymphocytes undergo apoptosis after brief exposure to UV light (34,35). In these experiments, we utilized the HPCG17 hybridoma, which undergoes a rapid metamorphosis through the programmed death pathway after UV irradiation. Within 24 h after irradiation, >90% of the cells became permeable to trypan blue and exhibited membrane blebbing, which indicates a loss of viability (Fig. 1). DNA isolated from cells at 2 h intervals after induction showed a typical apoptotic DNA ladder of 200 bp increments (Fig. 2). The fragments first appeared on an ethidium bromide stained agarose gel at the 2 h time point, increased in intensity for the first 8 h, and then remained constant. To ascertain that this was not a purely UV affected result, cells were also induced to undergo apoptosis by incubation in serum-free medium. A similar pattern of apoptosis was observed over a 24 h period in these cells (data not shown).


Figure 2. Degradation of high molecular weight DNA into an oligonucleosomal ladder over time after induction of apoptosis. 100 ng of DNA was isolated from HPCG17 cells at 2 h intervals after UV irradiation, run on a 0.8% agarose gel and stained with ethidium bromide. M, marker lane of [lambda] HindIII fragments; size in kb.

DNA fragmentation occurs at all stages of cell cycle

To determine when hybridoma cells undergo apoptosis during the cell cycle, irradiated cells were stained with propidium iodide and sorted on the basis of DNA content by flow cytometry. If fragmentation occurs preferentially in certain stages of the cell cycle, then the profile should change over time as the DNA undergoes fragmentation and the nuclei are extruded as blebs from the cells. As shown in Table 1, the cell cycle profile does not alter over time. This is consistent with the notion that fragmentation of DNA is not preferentially associated with any particular stage of the cell cycle. This supports earlier work, which demonstrated that UV irradiation stalls cells at multiple points in the cell cycle (41) and that apoptosis can occur in each phase of the cell cycle (42).

Table 1. Cell cycle profile after UV irradiation
Hours after irradiation Cell cycle stage (%)a Apoptotic cells (%)
G1 G2/M S
0 33.7 8.5 57.8 0
4 31.9 6.9 61.2 21
8 40.2 5.5 54.2 42
12 33.2 6.3 60.5 69
aDNA content was determined by measuring propidium iodide levels in HPCG17 cells using a cell sorter. Cell cycle was extrapolated from DNA content and cells were judged to be apoptotic if they had less DNA than cells in G1 phase.

DNA degrades at same rate regardless of transcriptional state or location

DNA was isolated at 2 h time intervals after UV irradiation and then tested by Southern blot analysis for degradation by hybridization with several probes which are summarized in Table 2. Three of the nuclear genes, the 28s ribosomal gene in the nucleolus, the [beta]-actin gene and the immunoglobulin [kappa] gene, are actively transcribed by hybridoma cells and are located in the euchromatin. The other two nuclear genes, C/EBP[alpha] and type II collagen, are not transcribed in lymphocytes (31,32) and are probably located in the more condensed heterochromatin. Mitochondrial DNA, which is not affected during apoptosis by the nucleases that cleave nuclear DNA (6), was used as a negative control to test for non-specific degradation of DNA during isolation. For the experiments, DNA from each time point was loaded onto the gel in triplicate so that a more accurate average could be determined. Degradation of the five nuclear genes was detected at 2 h after irradiation and continued until 8 h (Fig. 3). There was little degradation between 10 and 24 h (data not shown). As shown in Figure 4, all of the nuclear genes were degraded at the same rate as judged by their parallel slopes and identical endpoints. As expected, the mitochondrial DNA did not degrade appreciably during the first 10 h.


Figure 3. Southern blot analysis of degradation of genes during apoptosis. Ten µg of BamHI-digested DNA were separated on a 0.8% agarose gel and probed for degradation. Experiments were performed in triplicate.

Table 2. Characteristics of gene-specific probes
Probe Transcription in lymphocytes Cellular location
28s ribosomal gene active nucleus (nucleolus)
[beta]-actin active nucleus
Ig[kappa] active nucleus
C/EBP[alpha] inactive nucleus
Type II collagen inactive nucleus
Mitochondria active mitochondria

DISCUSSION

There is precedence for a hierarchy of gene accessibility to enzymes based on their transcriptional status. In DNA repair, transcribing genes are repaired faster than non-transcribing genes, and this difference is likely to be associated with chromatin structure within the euchromatin and heterochromatin (37). Thus, accessibility of a gene may be critical for an enzyme-related process to function. During apoptosis, one report has suggested that the degradation of DNA during apoptosis is non-random (36). Oligonucleosomal fragments were cloned and sequenced from apoptotic rat chloroleukaemia cells, and a higher than expected frequency of both long and short interspersed nuclear retroelements (LINES and SINES), and short, repetitive non-coding sequences were found. While this supports the theory that there is heterogeneity in the rate of degradation of nuclear DNA based on its accessibility to the endonucleolytic cleavage machinery, the relative rates of degradation of different parts of the genome have never been directly tested.

In earlier reports, oligonucleosomal ladders began to appear in ethidium bromide-stained gels at 60-90 min after the induction of apoptosis in cells and at 45 min after induction of apoptosis in a cell-free system (12,38-40). We report here that Southern analysis of DNA from a B cell hybridoma undergoing apoptosis indicates that degradation into oligonucleosomal fragments begins earlier than previously reported and has a definite endpoint. Previous detection of oligonucleosomal ladders was performed by visual inspection of ethidium bromide stained agarose gels (11,12,15,38). In this report we used the Southern blot approach, which greatly enhances the sensitivity of detection, and showed a clear and reproducible 15-30% loss of specific genes at the 2 h time point (Fig. 4). The linearity of the rate of degradation of the DNA from 0 to 8 h suggests that degradation starts immediately after the apoptotic signal is received and occurs concurrent with the fragmentation into large fragments. The sudden end of degradation of all loci at 8 h, even though 30% of the DNA remained uncut, suggests that there is a cut-off switch to the mechanism. One possible switch could be the escape of necessary factors such as Ca2+ or Mg2+ due to the increasing permeability of the cell membrane. Furthermore, the endonucleases responsible for cutting the DNA between nucleosomes do not discriminate between transcribing and non-transcribing genes, indicating there is no hierarchy of degradation. The evidence reported here supports the idea of random degradation of the DNA, regardless of transcriptional state or location in the nucleolus, euchromatin or heterochromatin.


Figure 4. Nuclear DNA degrades at the same rate during apoptosis regardless of transcriptional state. Southern blots from Figure 3 were quantified by a PhosphorImager, and the percentage of DNA remaining at each time point was calculated. Probes are marked as follows: +, mitochondrial DNA; [diamond], 28s ribosomal gene; [cone], [beta]-actin; [triangle], Ig[kappa]; [square], C/EBP[alpha]; and [circle], type II collagen.

Although the accessibility of enzymes to different parts of the genome may vary depending upon the transcriptional state of the locus or its location in the nucleus (37), in apoptosis, there is no sequential pattern of degradation. This may be due to the formation of DNA rosettes and subsequent single loops which makes normally cryptic areas such as the heterochromatin highly accessible to internucleosomal cutting (7,8,10-12). Alternatively, there may be changes in chromatin structures that take place when a cell is destined to die that renders the chromatin structure more accessible to endonucleases. The non-discriminatory pattern of cleavage would ensure that the entire DNA is rapidly degraded once the cell activates its destruction program.

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