A hydroxylapatite batch assay for quantitation of cellular DNA damage

ANALYTICAL

BIOCHEMISTRY

97, 77-84 (1979)

A Hydroxylapatite

Batch Assay for Quantitation DNA Damage1

PETER M. KANTER,~ AND HERBERT

of Cellular

S. SCHWARTZ

Department of Experimental Therapeutics, Grace Cancer Drug Department of Health, Rowe11 Park Memorial Institute, Buffalo, Nebcs York 14263

Center, New York 666 Elm Street,

State

Received August 31, 1978 A batch elution procedure is described for quantitative measurement of DNA damage. The technique is based on alkaline unwinding of cellular DNA and separation of singlestranded from duplex forms by step elution from hydroxylapatite with phosphate formamide. The method is rapid, permits large numbers of samples to be handled simultaneously, and consistently yields recoveries of >95% of total chromatographed DNA. Because as many as 1 x 10’ cells per batch may be analyzed, quantitation of the eluted DNA by nonradioactive methods is feasible. The method is standardized with respect to the unwinding constant p, the alkaline DNA unwinding unit Mn,, and the DNA-damaging efficiency of ionizing irradiation.

Rydberg in 1975 (1) described a sensitive method for estimation of DNA damage in mammalian cells. The method was based upon the observations of Ahnstrom and Erixon (2) who showed that alkaline transformation of duplex mammalian DNA to the single-stranded form occurs slowly and uniformly, but is accelerated after cell exposure to low doses of ionizing irradiation. Rydberg (l), using the time-dependent alkaline denaturation and column chromatographic separation of single-stranded from duplex DNA on hydroxylapatite gels at elevated temperatures (6O”C), established the theoretical background for estimating DNA damage. Duplex unwinding begins at the end of each DNA unit, frank strand break, or alkaline-labile region. Denaturation proceeds progressively, and each unwinding point present or introduced in DNA

increases the amount of single-stranded DNA formed during the limited period of alkaline denaturation. We have adapted the basic concept to a batch elution procedure for chromatography of mammalian DNA. The new procedure has been simplified for routine assays and has been scaled up to handle large numbers of cells, thereby permitting DNA quantitation by the nonradioactive fluorescent method of Kissane and Robins (3). MATERIALS

AND METHODS

Hydroxylapatite (Bio-Gel HTP, DNA grade) was purchased from Bio-Rad (Richmond, Calif.). Reagent grade formamide and the dihydrochloride salt of diaminobenzoic acid were obtained from Eastman (Rochester, N. Y.). Gradient grade CsCl was obtained from Schwarz/Mann (Orangeburg, N. Y.). ACS scintillation cocktail and [2-14C]thymidine (TdR) (45 mCi/mmol) were purchased from Amersham Searle (Arlington Heights, Ill.). Radioactive samples were counted in polyethylene scintilla-

’ Supported in part by the National Cancer Institute, USPHS Grants CA-14750 and CA-02301. 2 In partial fulfillment of the requirements for a Ph.D. in the Department of Pharmacology, Roswell Park Memorial Institute Division of the Graduate School, State University of New York at Buffalo. 77

0003-2697/79/110077-08$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

78

KANTER

AND SCHWARTZ

tion vials with a Packard 3320 liquid scintillation counter. Dialyzed fetal calf serum, RPM1 1640 media, and penicillin-streptomycin (10,000 units penicillin/ml, 10,000 pg streptomycin/ml) were obtained from Grand Island Biological Company (Grand Island, N. Y.). Cell culturing and labeling. CCRF-CEM cells, a human acute leukemia line (4), and P288 and EL4 cells, carcinogen-induced lymphomas of DBA/2 and C57Bl mice, respectively, were maintained in log growth in RPM1 1640 culture media supplemented with 10% heat-inactivated (56°C 30 min) dialyzed fetal calf serum and 1% penicillinstreptomycin solution. Cells were labeled for 18 h with 0.1 FCi [14C]TdR/ml, centrifuged, and resuspended in fresh medium for 1 h before use. Irradiation. Leukemia cells were centrifuged, resuspended in 0.1 N Na phosphatebuffered (pH 7.4) 0.9% NaCl solution (PBS)3 to concentrations of 5 x 105- 1 x 10’ cells/ml, and 1.O-ml aliquots were pipetted into glass vials and left uncapped. The cells on ice (air atmosphere) were irradiated with a GE Maxitron 300 therapy machine, using settings of 20 mA, 300 KVP, a f/4 mm Cu filter, and a target-to-radiation-source distance of 50 cm. Irradiation rate was 200 radlmin. Cell lysis. Cells were lysed in vials with an equal volume (1 ml) of 0.1 N NaOH forcefully expelled from a manual pipetter. During the entire lysis period (30 or 60 min at 2 I-22°C) care was taken to protect samples from light and to minimize vibration of the vials. At the end of the lysis period, 1 ml of 0.1 N HCI was pipetted into each vial, and the vials were shaken to ensure prompt and thorough neutralization (phenol red indicator). Sodium lauryl sarcosinate (1 ml of a 2% solution containing 0.02 M Na EDTA, pH 7.0) was then added to the vials, the DNA was sheared by six rapid passages 3 Abbreviations used: PBS, 0.1 N Na phosphatebuffered, pH 7.4, 0.9% NaCl solution; [14C]TdR, [2JV]thymidine.

through a 22-gauge needle, and the lysate was stored at 4°C until chromatography. Refrigerated lysates were stable for at least 6 weeks. Chromatography. Hydroxylapatite (0.5 g) was brought to a boil in 5 ml of 0.01 M potassium phosphate buffer, pH 7.0, pipetted into 15-ml screw-top test tubes, and centrifuged for about 15 s at 600g; the supernatant was discarded, and the tubes were placed in a 60°C constant-temperature bath. Formamide to a tinal concentration of 10% was added to cell lysates and the mix was transferred to the tubes containing boiled hydroxylapatite and vortexed briefly. After 15 min at 60°C with occasional brief vortexing, tubes were centrifuged as above; supematants were discarded, and 5 ml of 0.01 M potassium phosphate buffer (pH 7.0) containing 20% formamide was added. After vortexing the tubes were again bathed at 60°C for 10 min with occasional mixing and then centrifuged as above, the tubes were returned to the bath, and the supernatant was discarded. Unincorporated nucleic acid precursors may be recovered quantitatively from this fraction. Singlestranded DNA was then selectively eluted from the gel by two successive IO-min incubations (60°C) with 5 ml of 0.125 M potassium phosphate buffer, pH 7.0, containing 20% formamide. As before, the tubes were kept in the bath at all times except for vortexing. Tubes were centrifuged as before and supematants collected (not combined). Duplex DNA was removed by two successive lo-min incubations with 5 ml of 0.5 M potassium phosphate buffer (pH 7.0) containing 20% formamide. Supematants were again collected after centrifugation. All recoveries of radioactive materials exceeded 95% unless indicated otherwise. If DNA was prelabeled ([14C]TdR), 1 .O-ml aliquots of the eluates were counted in plastic scintillation vials containing 18 ml of scintillation fluid and 1 ml of 1.0 N HCl. DNA in eluates was assayed by a modified fluorescent method of Kissane and

QUANTITATION

OF CELLULAR

Robins (3). DNA (l.O-ml aliquots) was coprecipitated with bovine serum albumin (0.1 ml of a 0.3% solution) in conical centrifuge tubes with 2 vol of 20% trichloroacetic acid overnight at 4°C. Tubes were centrifuged at 600g for 15 min at 0°C. Supernatants were discarded, pellets were extracted with 0.5 ml of 0.1 M potassium acetate in ethanol for 15 min on ice, recentrifuged for 5 min at 600g (o”C), and the supernatants were discarded. The pellets were then extracted with 0.5 ml absolute ethanol (30 min; 6o”C), centrifuged for 15 min at 600g (20”(Z), supernatants were discarded, and the pellets were dried overnight at room temperature or for 2 h in a drying oven (60°C). The dihydrochloride salt of diaminobenzoic acid (25 ~1, 2.0 M) was added to each tube, the tubes were stoppered and heated for 45 min at 60°C. Fluorescent products were dissolved in 0.5 ml of 0.6 N perchloric acid, and analyzed in quartz cuvettes (7 x 7 x 35 mm) with an Aminco Bowman spectrofluorometer at an excitation wavelength of 420 nm, and an emission wavelength of 520 nm. Zsopycnic centrifugation. Eluates analyzed by isopycnic CsCl centrifugation were dialyzed at 4°C for 24 h against four changes of PBS. CsCl(2.58 g) was dissolved in 2.0 ml of dialyzed material (refractive indices were adjusted to 1.40) and centrifuged for 44 h at 30,000 rpm (Beckman L5-50; SW50.1; 20°C). Fractions (0.1 ml) were taken from the bottom of the centrifuge tubes and the refractive index was determined in aliquots (10 ~1) from alternate fractions. Radioactivity in each fraction was measured after addition of 0.1 ml distilled water and 5 ml scintillation fluid to each fraction. RESULTS

In preliminary of DNA samples perature with a of formamide in

experiments batch elution was tested at room temhigh concentration (50%) the elution buffers as sug-

19

DNA DAMAGE

20

l0L, 0

IO

20

30

40

50

% FORMAMIDE

FIG. 1. Total DNA elution recovery (0) and duplex DNA (as percentage of total DNA eluted) (0) with various concentrations of formamide in the elution buffers. Prelabeled ([2-W]TdR) P288 leukemia cells (5 x 106; 4.3 x lo5 cpm) were lysed in alkali, and chromatographed by procedures outlined in Results.

gested by studies with bacterial and viral (5,6) DNA. When DNA from x-irradiated CCRF-CEM cells was chromatographed using similar conditions, the results indicated that elution of single-stranded DNA was incomplete and that recoveries of total DNA from the hydroxylapatite gel dropped progressively with increased doses of irradiation. Figure 1 illustrates total recoveries and percentages of DNA eluted in the doublestranded form from untreated cells after various concentrations of formamide were added to the elution buffers. The elution temperature was 60°C but to test suboptima1 handling conditions (i.e., a temperature drop during centrifugation of a large number of samples) the tubes were kept at room temperature for a 5-min period after each centrifugation. When no formamide was present in the buffer, most of the DNA was eluted by the 0.125 M phosphate buffer washes, with unsatisfactory recoveries (84%). Increasing formamide to a concentration of 20% improved recovery (98%) with an increase in the amount of DNA eluted in the duplex form. At higher con-

80

KANTER

AND SCHWARTZ

centrations of formamide (30, 40, 50%) the relative proportion of duplex DNA was decreased, with a concurrent loss of total DNA recovered. The optimal concentration of formamide for routine use was 20%, as this gave the highest recoveries under the adverse conditions of extensive sample cooling which may occur when large numbers of samples are eluted concurrently. To evaluate fidelity of the eluates, lysates of prelabeled cells irradiated with 1000 rad were eluted from hydroxylapatite with three extractions with 0.125 M phosphate buffer followed by three extractions with

4OOc

0.5 M phosphate buffer (60°C 20% formamide). Aliquots from each extraction were dialyzed against PBS followed by isopycnic centrifugation in CsCI. The results (Fig. 2) indicate that the first two extracts (A and B) with 0.125 M phosphate buffer contain single-stranded DNA (buoyant density 1.709- 1.710), and that the three with 0.5 M phosphate (D, E, and F) contain doublestranded DNA (buoyant density 1.6901.693). The asymmetric peak in fraction C demonstrates a shoulder in the doublestranded region, indicating the third 0.125 M phosphate extraction eluted small amounts

I 710

A 4,

3500 ,I

3OOC 2500

0

2000 1500 1000 500 cl 600 k 400 300 200 too 0 200 100 0

I F

300

5

IO

G93 I.710

15 20 25 5 IO 15 20 25 FRACTION NUMBER

FIG. 2. Isopycnic cesium chloride centrifugation TdR tumor cells (CCRF-CEM, 5 x 10”) irradiated DNA eluted sequentially with 0.125 M phosphate elution with 0.5 M phosphate buffers. Procedures

analysis of eluates after chromatography of [2-l%]with 1000 rad. Panels A, B, and C are profiles of buffer; panels D, E, and F are the profiles after were as outlined under Methods.

QUANTITATION

OF CELLULAR

DNA DAMAGE

81

lysis times was also demonstrated with CCRF-CEM cells (Fig. 4). In addition to DNA quantitation by standard scintillation a70 s technique, aliquots of the eluates were s 60 assayed for DNA by the fluorescent method 350 8 of Kissane and Robins (3) as described in 40 the methods section. The results obtained by these two methods (Fig. 4) were in close agreement, and indicated that the DNA 300 600 900 RAD strand damage caused by as little as 100 rad of x irradiation can be detected in unlabeled FIG. 3. Percentage of DNA recovered in the duplex form after various doses of x irradiation. Leukemia mammalian cells. cells were prelabeled for 18 h with 0.1 &I [2-W]The effect of cell number on the estimaTdRml, washed, irradiated on ice, and lysed in dilute tion of DNA damage is shown in Table 1. alkali. All elution buffers contained 20% formamide. cells (5 In all cases recovery of radioactive DNA (>l x lo5 Aliquots (1.0 ml) of CCRF-CEM cpm) exceeded 95%. (0) P288 leukemia cells (5 x 106) x 105- 1 x 10’ cells/ml) were lysed in alkali lysed for 60 min and chromatographed with 0.125 M (0.05 N NaOH, final) for a 60-min period, PO, (two elutions) and 0.5 M PO, (two elutions); (A) and the lysates were sheared, neutralized, EL4 leukemia cells (5 x 106) lysed for 30 min and and chromatographed as outlined in the chromatographed with two successive elutions of methods section. In all cases total DNA 0.125 M PO, followed by two successive elutions recoveries from the gel were similar, and with 0.5 M PO, buffer (x) or three successive elutions with each buffer (0). >95%. A small drop in the proportion of duplex DNA (0.881-0.832, unirradiated of duplex DNA, estimated to be 1% of the cells) after lysis and chromatography of total DNA. decreasing numbers of cells may reflect a Figure 3 shows results after irradiation and change in the unwinding constant /3 due to DNA chromatography of murine leukemia DNA entanglement and gel formation when cells p288 and EL4 prelabeled with [14C]TdR. very high cell numbers are used. A similar Both cell lines after 30-min lysis have similar pattern was evident in results obtained after decreases in the proportion of DNA eluted lysis and chromatography of irradiated in the double-stranded form with increasing (1000 rad, 0°C air atmosphere) cells. Difdoses of x irradiation. The ratio of doublestranded DNA to total is almost identical 90 (Fig. 3) after two or three successive elu2 00 tions of P288 DNA with 0.125 and 0.5 M ; 70 phosphate. The gain in recovery from the third extraction with each buffer is about 8 . 2%, indicating that two successive extrac50 tions for most assay procedures are ade2 60 II-;. I quate. Prolonging time for cell lysis and 300 600 900 1200 DNA unwinding from 30 to 60 min (P288, RAD Fig. 3) results in a decreased amount of FIG. 4. Percentage of DNA recovered in the duplex duplex DNA in both unirradiated (control) form after various doses of x irradiation. CCRF-CEM (from 92 to 86%; recoveries >95%) and leukemia cells (5 x 10”) were prelabeled for 24 h with 0.1 &i/ml [2WZ]TdR, washed, irradiated on experimental cells. The rates of unwinding ice, and lysed for 30 min (0) or 60 min (0) with DNA in both control and irradiated (~1200 rad) quantitation by standard scintillation technique or by is log linear with respect to tB (see Discussion). the fluorescent method of Kissane and Robins (X), The change in duplex DNA with prolonged 60-min lysis. 90 60

82

KANTER TABLE EFFECT

AND SCHWARTZ

1

OF CELt NUMBER ON DNA DAMAGE ESTIMATNN

F CellNo. 1x 5x Ix 5x

107 106 108 105

Control

0.881 0.880 0.862 0.832

1000 rd

0.431 0.436 0.39Q 0.316

n

tween unwinding points and /3 is a constant less than 1. To determine p, it is assumed that IS&V and K are constant during alkaline treatment of uni~adiated cells and that p is then a function of F and t for unwinding for 30 and 60 min:

5.6 5.5 5.3 5.3

u Preiabeled ([2-L*CfTdR) CCRF-CEM cells were cam&rated to 1 x I@ cells/ml PBS, irradiated (loo0 rad), and lysed after dilution to the cell numbers indicated in the table. F is the fraction of DNA eiuted in the duplex form, and n is the number of breaks per DNA unit (Mn,) in irradiated cells calculated in terms of unirradiated celts similarly lysed and chromatographed (see Discussion).

ferences in the calculated number of breaks (n) per DNA unwinding unit (Mn,; see Discussion), however, did not vary significantly.

From P288 and CCRF-CEM cell cultures, the proportion of double-stranded DNA (F) was 0.92 t 0.01 at 30 tin and 0.88 & 0.01 at 60 min for both lines. The unwinding constant was calculated to be 0.62, which is in agreement with the values found by Rydberg (1) at 20-21°C; 0.64, 0.67. The number of unwinding points (p) per alkaline unending unit of DNA after irradiation is obtained from Eq. [l]: Mn,

* In F. = Mnx 1 In F,,

[31

and

Hydroxylapatite chromatography by batch elution can be used to measure low levels of mammalian DNA damage induced by irradiation. The method is rapid, reliable, and readily adaptable to routine assays. Formamide in the elution buffers and the packing characteristics of the gel permits handling large numbers of samples by centrifugation, and as many as 48 samples can be assayed concurrently. Rydberg (1) derived the relationship between strand separation of duplex DNA in alkali where randomly distributed breaks are introduced:

M&J p=Mn,=-’

In Fx In F0

t41

where subscripts x and 0 are irradiated and unirradiated samples, respectively. The number of breaks (n) per unit DNA is: n=p-1.

PI

The values of p, n , and nirad (breaks/unit DNA/rad) obtained by batch elution are shown in Table 2 with P288 and CCRF-CEM cells after unwinding in alkali for 30 and 60 min each. For comparison, n and nirad (CCRF-CEM celfs, 30 min lysis) were estimated using column chromatography as described by Rydberg (1); these results agreed within experimental error with those 1n F = 5 *tfl, III obtained by batch elution. Mn Mno, the size of the alkaline DNA unwinding unit, may be estimated from the where F is the fraction of double-stranded irradiation data. Extrapolation of the linear DNA remaining after alkaline denaturation portion of the curves in Figs, 3 and 4 yields for time (t), and K is an assumed constant for rotational and frictional forces. Mn is a D3, value of 1475 t 287 rad. By using the num~r-average molecular weight be- the formula suggested by Kohn and Grimek-

QUANTITATION

OF CELLULAR

83

DNA DAMAGE TABLE

Ewig (7): 5 x lo’* daltons/cell 8 breaks/celYrad *OS7 ’ the number average molecular weight of the alkaline unwinding unit is 4.2 x 108, a value in close agreement to those obtained by alkaline sedimentation studies (1 . 1- 5.4 x lo*) (8-10). In Table 2 the number of breaks (or alkaline-labile lesions) per DNA unwinding unit per rad averaged 5.6 x lop3 for CCRF-CEM and 6.3 x 10e3 for P288 cells. Using 6 x 10u3 breaks as the approximate value, the efficiency of irradiation (11) in both cell lines is approximately 1.2 x lo-” breaks/daltons/ rad, equivalent to 52 eV/break, a value midrange (36-70 eV/lesion) for those found by others in mammalian cells (1 l- 14). Various technical considerations must be closely followed to insure reliable and reproducible results. Excessive vibration of samples during the period of alkaline unwinding results in accelerated rates of denaturation. The pH of the washing and eluting buffers are critical; deviation from pH 7.0 results in decreased elution of singlestranded DNA from the gel, leading to unreliable values of F and decreased total DNA recovery. Retention by hydroxylapatite of duplex DNA during elution of the single-stranded DNA with 0.125 M phosphate buffer may be impaired with older gels. We therefore do not use hydroxylapatite which has been stored (4°C) for more than 6 months without a new determination of the concentration of phosphate buffer that promotes quantitative elution of bound single-strand DNA without release of duplex DNA. In our experience phosphate concentrations of 0.105 to 0.120 M may be required with such-aged hydroxylapatite. In our hands prelabeled untreated (control) cells of high viability (95% trypan blue exclusion) lysed for a 60-min period yield 80-88% intact DNA after chromatography (dependent partially upon cell number in the lysate).

2

RADIATION-INDUCED DNA DAMAGE IN CCRF-CEM AND n88 CELLS” nlrad Cells CCRF-CEM

p288

Rad

t

F

n

0 100 300 600 900 1200

30 30 30 30 30 30

0.92 0.88 0.79 0.67 0.59 0.54

0 100 300 600 900

60 60 60 60 60

0.88 0.81 0.71 0.61 0.51

0 0.54 (0.44) 1.83 (1.72) 3.81 (4.20) 5.33 (6.00) 6.41 (7.20) av 2 SD = (av 2 SD = 0 0.65 1.67 2.86 4.26 av t SD =

0 100 300 900

30 30 30 30

0.92 0.86 0.80 0.54

0 0.81 1.68 6.41

0 100 300 900

60 60 60 60

0.86 0.78 0.66 0.44

0 0.65 1.75 4.44

(XlW

5.4 (4.4) 6.1 (5.7) 6.3 (7.0) 5.9 (6.6) 5.3 (6.0) 5.8 2 0.4 5.9 ? 0.9) 6.5 5.6 4.8 4.7 5.4 -c 0.7 -

8.1 5.6 7.1 av = 6.9 6.5 5.8 4.9 av = 5.7

cIPrelabeled ([2-‘*C]TdR) CCRF-CEM and P288 leukemia ceils were irradiated, lysed, and chromatographed as outlined in legends of Figs. 3 and 4. The symbol t represents alkaline unwinding time, F the fraction of DNA eluted in duplex form, and n the number of breaks per DNA unit. Values in parentheses were determined by column chromatography as described by Rydberg (1).

If cells of poor viability are used, lower F values may result with a greater variability in the data obtained in DNA damage studies. Other means currently in use to estimate DNA damage include alkaline sucrose gradients (15), antinucleoside antibodies (16)) alkaline elution (7), and part&l enzymatic degradation of DNA by Sl nuclease (17). The alkaline sucrose gradient method, first described for bacterial cells by McGrath and Williams (15) and also described for

84

KANTER

AND SCHWARTZ

unlabeled mammalian cells (8), depends upon cell lysis and complete denaturation of the DNA in alkali prior to centrifugation; the latter is particularly difficult to assess because it must proceed to completion which requires varying conditions for different cell types. The technique is expensive and only accurate when small numbers of cells are used; it is not well adapted to large numbers of runs on a routine basis and cannot accurately quantitate DNA damage produced by low levels of ionizing irradiation (< 1000 rad). Kohn and Grimek-Ewig (7) described a technique of alkaline elution of DNA from cells lysed on cellulose triacetate filters. The method appears to be adaptable to routine assays, and sufficient cells may be processed so that nonradioactive DNA determinations are feasible (19). The technique requires extensive sample numbers to accurately define elution patterns and has variable recovery losses due to DNA retention on the filters. The method is sensitive, and as few as 50 rad of x-irradiationinduced DNA damage can be quantitated. Batch elution of DNA from hydroxylapatite has been standardized with respect to p (the unwinding constant) and Mn,, (the alkaline unwinding unit); similarly, the calculated values for the efficiency of irradiation (eV/DNA break or break&ad) are in acceptable agreement with values obtained by others using different techniques (11,15,16,19) or as shown here (Fig. 3) by column chromatography. In addition to utility in quantitation of DNA damage by ionizing irradiation, the described hydroxylapatite batch assay may be useful in studies involving certain chemical agents that react with DNA. A manu-

script describing this application DNA studies is in preparation.

in drug-

ACKNOWLEDGMENTS We gratefully acknowledge the assistance of I. W. H arper in the preparation of this manuscript.

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