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Measurement of DNA damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay
ANALYTICAL
BIOCHEMISTRY
106,169-174
(1980)
Measurement of DNA Damage in Unlabeled Mammalian Cells Analyzed by Alkaline Elution and a Fluorometric DNA Assay LEONARD C. ERICKSON,‘RAINHARDT OSIEKA,~ NANCY A. SHARKEY, ANDKURT W. KOHN Laboratory
of Molecular Pharmacology, National Cancer Institute,
Developmental Therapeutics National Institutes of Healih,
Program, Bethesda,
Division of Cancer Maryland 20205
Treatment,
Received January 25, 1980 A fluorometric procedure is described that can be used in the alkaline elution technique for the measurement of DNA damage in cells whose DNA is not, or cannot be, radioactively labeled. The procedure can be used for the measurement of DNA single-strand breaks, DNA-protein crosslinking, and DNA interstrand crosslinking, and possibly other DNA lesions produced in unlabeled cells. Although developed for the measurement of DNA damage in tissue-cultured cells, the technique is applicable to the measurement of DNA damage in cells isolated from tissues exposed to DNA damaging agents in vivo.
The alkaline elution technique, developed in this laboratory (1 - 3), has proven to be a powerful and sensitive method for the measurement of a variety of DNA lesions produced in mammalian cells by various types of DNA-damaging agents. The alkaline elution method measures damage in the DNA of cells which have been immobilized on a membrane filter, and lysed with detergent. Following lysis, an alkaline solution of pH 12.1 is slowly pumped through the filter. DNA is eluted from the filter as a function of molecular weight, with lowmolecular-weight DNA eluting rapidly, and high-molecular-weight DNA eluting at later times. Among the lesions measured by alkaline elution are DNA single-strand breaks (4- 13), DNA-protein crosslinks (14- 19), DNA interstrand crosslinks (16,17,19,20-24), protein-concealed single-strand breaks (25,26), and DNA double-strand breaks (27). The alkaline elution technique has 1 To whom requests for reprints should be addressed at Building 37, Room 5D17, National Institutes of Health, Bethesda, Md. 20205. * The Innere Klinik (Tumorforschung) Universitaetsklinikum Essen, Federal Republic of Germany. 169
also proven useful for the isolation of DNA replication intermediates (28). In all of the referenced studies it was necessary to prelabel the cellular DNA with [‘“Cl- or [3H]thymidine prior to alkaline elution analysis. Recently several laboratories, including our own, have attempted to measure DNA singlestrand breaks by fluorometric analysis of eluted DNA (9,13). However, no fully documented procedure has been published, testing the completeness of DNA recoveries and the validity of the assays. We now report a convenient and accurate fluorometric procedure applicable to unlabeled cells and tissues analyzed by the alkaline elution technique. MATERIALS
AND METHODS
Cell culture. L1210 mouse leukemia cells were maintained in suspension culture in RPM1 1630 medium, supplemented with 20% heat-inactivated fetal calf serum (Flow Laboratories, Rockville, Md.), penicillin, and streptomycin. Cultures were maintained in exponential growth phase with a doubling time of about 12 h. Cells in experiments 0003-2697/80/l 10169~06$02.00/0 Copyright All rights
0 1980 by Academic Rcss, Inc. of reproduction in any form reserved.
170
ERICKSON
were used either unlabeled, or the DNA was labeled with [2-14C]thymidine (>50 mCi/mM, New England Nuclear, Boston, Mass.). Labeled cells were grown for 20 h in either 0.02 pCi/ml [14C]thymidine, or 0.2 pCi/ml [14C]thymidine as indicated in the text. HN23 was obtained from the Drug Development Branch, National Cancer Institute, NIH, Bethesda, Maryland. The drug was dissolved in 0.1 N HCl and stock solutions were frozen at -20°C. The drug was added to cultures of L1210 cells at a concentration of 1.0 x IO6 cells/ml in 1630 medium containing 20% fetal calf serum. Drug exposures were terminated by two centrifugations at SOOg for 5 min and resuspension in fresh 1630 medium. Alkaline elution. The alkaline elution procedure used has been described in detail in several recent publications (2,3). For the present experiments following drug treatment, cells were chilled on ice in 1630 medium, irradiated with 300-R X rays, and analyzed by alkaline elution. X irradiation was delivered by two Philips RT-250 X-ray tubes operating at 200 KeV and 15 mA, with 0.25-mm copper and 0.55~mm aluminum filters. Following irradiation, the cells were resuspended in 15 ml ice-cold PBS, and deposited by mild suction on a 2.0-km-poresize polyvinyl chloride (PVC) filter (Millipore Type BSWP 02500, Millipore Corp., Bedford, Mass.). The cells were immediately lysed with 5 ml of a solution of 2% sodium lauryl sulfate (SDS), 0.02 M EDTA, 0.1 M glycine (pH 10.0) which was allowed to drip through the filter by gravity. The lysate was deproteinized by pumping lysing solution containing 0.5 mg/ml proteinase-K (E. M. Laboratory, Elmsford N. Y.) through the filter at 0.035 ml/min for 30 min. The remaining proteinase solution was gently 3 Abbreviations used: HN2, bis(2-chloroethyl)methylamine; PBS, phosphate-buffered saline, 0.15 M NaW0.014 M KH,POd0.086 M K,HPOI; DABA, 35 diaminobenzoic acid, SDS, sodium lauryl sulfate.
ET AL.
aspirated off, and a solution containing 0.02 M EDTA, 0.1% SDS, and tetrapropylammonium hydroxide to yield a pH of 12.1, was added and pumped through the filter at 0.035 mYmin. Five 3-h (6.3-m]) fractions were collected into scintillation vials. At the end of the elution, the remaining solution was decanted off the filter, and the filter removed to a clean scintillation vial and air dried. A 5-ml wash of tetrapropylammonium hydroxide was pumped through the filter holder and pump lines, and was collected and processed as the sixth fraction. Sample processing and fluorescence measurement. In the standard alkaline elution assay, since the DNA is radioactively labeled, samples are processed by the addition of a liquid scintillation cocktail to the eluted fractions, and counting in a liquid scintillation counter. For the fluorometric analysis, DNA in the eluted fractions must be quantitatively and reproducibly recovered from the eluting solution. DNA was recovered by the addition of 2 vol of 95% ethanol containing 0.3 M sodium acetate (high purity, high-pressure liquid chromatography grade, Fisher Scientific, Pittsburgh, Pa.), and 0.055 M glacial acetic acid. The samples were capped, mixed, and chilled to -20°C for 30 min. The precipitated DNA in each fraction was trapped on a 0.2+mpore-size Durapore filter (type GVWP 02500, Millipore Corp.) using a 30-position Millipore sampling manifold. Vials and filters were washed with a 50% ethanol solution, the filters removed to clean scintillation vials, and air dried. Following air drying, all filters were processed by a modification of the fluorometric technique of Kissane and Robbins (29). A 2 M DABA solution (3,5-diaminobenzoic acid dihydrochloride, Aldrich Chemicals, Milwaukee, Wise.) was prepared with glass-distilled water, shaken with 50 mg/ml activated charcoal (Sigma, St. Louis, MO.), and filtered through a 0.2~pm-poresize Millipore Sterifil D-GS filtration unit.
MEASUREMENT
OF DNA DAMAGE
DABA solution, 400 ~1, was added to each filter (eluted fraction precipitate, and PVC elution filter), the vials were tightly capped with polyethylene-lined caps, and heated at 60°C for 45 min. Following heating, 1.6 ml 1 N HCl was added to each vial, the vials were shaken vigorously, and the fluorescence was determined. Three Durapore and three PVC blank filters were prepared for each experiment by running the appropriate solutions through them. This was necessary to accurately establish the background fluorescence for each filter. Additionally, a standard curve of precipitated DNA (O-10 pg) was routinely prepared for each experiment. Fluorescence was read in a Farrand Ratio-2 fluorometer (FOCI Corp., Valhalla, N. Y .) equipped with two 405nm interference filters in series for excitation wavelengths, and two 525nm interference filters for emission readout. Samples were pumped into a flow cuvette using a peristaltic pump, and recovered for scintillation counting by reversal of the peristaltic pump. In this way fluorescence and radioactivity could be determined in the same sample. For liquid scintillation counting, 10 ml Aquassure Liquid scintillation cocktail (New England Nuclear) was added to each sample, and the samples were counted in a Packard 2450B liquid scintillation counter. The DABA/ Aquassure cocktail is highly quenched, however, carbon-14 can be conveniently counted using an open tritium window at an efficiency of approximately 50%. RESULTS AND DISCUSSION
To accurately measure DNA damage by alkaline elution, DNA must be quantitatively and reproducibly recovered from the eluted fractions and the elution filters. The data presented in this study demonstrate that these criteria can be met using the procedure as described. After testing many types of filters which were unacceptable in their DNA trapping ability, wetability, or compatability with
IN UNLABELED
MAMMALIAN
CELLS
171
CPM IN 1 ml ALIQUOT OF DABA SOLUTION lx 10’1
FIG. 1, Extraction of precipitated W-labeled DNA from Durapore filters (see Materials and Methods). Following incubation with DABA and addition of HCl as described under Materials and Methods (total volume 2 ml), 1 ml of DABA solution was removed, a blank Durapore filter added, and the r4C counts per minute were determined in a liquid scintillation counter. The counts per minute observed are plotted versus the counts per minute in the remaining 1 ml of DABA solution containing the original precipitation filter. Equivalence is indicated by the dashed diagonal line. Three replicate determinations are plotted for the following DNA concentrations: 2.5,5,7.5, and 1Opgof added DNA.
DABA, we chose O.Zpm-pore-size Durapore filters (Millipore, type GVWP) to recover DNA in the eluted fractions. Studies with radioactive DNA demonstrated a trapping efficiency of better than 98% for DNA precipitated out of pH 12.1 tetrapropylammonium hydroxide, when mixed with 2 vol of ethanol/sodium acetate as described under Materials and Methods. When known amounts of unlabeled calf thymus DNA were precipitated onto Durapore filters from the eluting solution, reacted with DABA, and read in a fluorometer, a highly reproducible linear response to DNA concentration was observed. A DNA concentration of 10 pg/sample yielded fluorometric readings of approximately 0.30 in our system. Although the trapping efficiency of the filter is very important in the recovery of the DNA from the eluted fractions, it is also necessary to show that the precipitated DNA can be accurately recovered from the
172
ERICKSON
t w 0.30 1 0.20 s2 0.10 0 0.00
l?Ll
2.5 CELLS
7.5 5.0 PER FILTER (x 10’)
10.0
FIG. 2. Determination of the amount of DNA retained on the 2.0-pm-pore-size PVC filter (Millipore, type BSWP) elution filter. The indicated number of “Clabeled L1210 cells was added to the filters, lysed as described under Materials and Methods, and tetrapropylammonium hydroxide (pH 12.1) was pumped through the filter for 1 h. The filters were removed from the funnels, air dried, reacted with DABA as described, and the fluorescence at 525 nm determined. Symbols indicate the mean k 1 SD for three replicate determinations.
precipitation filter. Figure 1 shows that DNA precipitated onto Durapore filters can be completely extracted from the filter by heating in 2 M DABA. A small quantity of high-specific-activity 14C-labeled L1210 DNA was added to unlabeled calf thymus DNA, precipitated, heated in 400 ~1 of DABA, and 1.6 ml 1 N HCI was added. The solution and filter were shaken vigorously and 1 ml of DABA/HCl solution was removed and added to a scintillation vial containing a blank Durapore filter. Ten milliliters of Aquassure LSC fluid were added to this vial and to the original vial with the precipitation filter plus the remaining 1 ml of DABA/HCl, and both vials were counted in a liquid scintillation counter. This test was performed in triplicate, with four concentrations of added DNA: 2.5, 5, 7.5, and 10 pg DNA. If the DNA had not been completely extracted from the Durapore filter, more of the 14C counts per minute should have been observed in the 1 ml DABA/HCl plus the original Durapore precipitation filter. Figure 1 shows that this was not the case. It can be seen that the
ET AL.
counts per minute in both samples of DAB& HCI are equivalent, indicating that recovery of DNA from the filter is complete. Equally as important is the quantitative and precise recovery of the DNA remaining on the PVC filter at the end of the elution. Figure 2 shows that the fluorescence observed following treatment of the PVC filter with DABA is in fact reproducible and proportional to the cell number added to the filter. This point is extremely critical, since the DNA remaining on the filter is a critical value in the assessment of DNA damage in treated cells. Figure 3 shows that, as was demonstrated from the Durapore filter in Fig. 1, the extraction of the DNA from the PVC filter is complete. These data were obtained by measuring the radioactivity remaining on the filter from 2.5, 5, 7.5, and 10 x lo6 14C-labeled L1210 cells added to the filter as described in the legends of Figs. 2 and 3. When the fluorescence obtained with ulfiltered DNA (air dried in a vial) was compared to the fluorescence of the same quantity of DNA precipitated onto a filter, we found approximately 50% quenching of the readings. This may be due to the variety of reagents run through the filters or the filter material
7.5 10.0 2.5 5.0 CPM IN 1 ml ALIQUOT OF DABA SOLUTION (x 10’) FIG. 3. Extraction of “C-labeled DNA from the PVC filters shown in Fig. 2. The protocol used was identical to that described in Fig. 1. Individual symbols indicate three replicate determinations for 2.5, 5, 7.5, and 10 x 10s cells used per filter. Diagonal dashed line indicates equivalence.
MEASUREMENT
OF DNA DAMAGE
itself. When the Durapore and PVC filters were compared for quench characteristics by air drying a DNA solution onto mock filters, the quenching of fluorescence by the two filters was virtually identical. Therefore, although some quenching of fluorescence occurs from the procedure, it is similar in both filter types so that no correction factor between the filters is necessary. For a fluorometric application of the alkaline elution technique to be useful it must give results qualitatively and quantitatively similar to the standard radioactive alkaline elution procedure. This is shown to be the case in Fig. 4, where the interstrand crosslinking produced by the anti-tumor agent HN2 (20,22) has been measured by fluorescence, and radioactivity in the same cells (upper panels). In this experiment 1.5 x lo6 14C-labeled cells were analyzed and the elution pattern determined by the fluorometric procedure is shown by solid symbols, and the elution pattern determined by radioactivity is shown by open symbols. To measure DNA interstrand crosslinking, the cells are irradiated with 300-R X rays. Interstrand crosslinking reduces the elution rate of DNA containing crosslinks. For this reason the 300 R only control pattern of the upper left panel is reproduced in the right panel for reference. These results demonstrate that the fluorometric analysis is in close aggreement with the radioactive analysis. Similar results were obtained with the anti-tumor agents cis-platinum(I1) diaminedichloride (23), and L-phenylalanine mustard (22), data not shown. The lower panels of Fig. 4 illustrate fluorometric elution assays of drug-treated unlabeled cells in which quantitation of interstrand crosslinking has been improved by the addition of a small number of untreated 14C-labeled cells as an internal reference. Because elution problems sometimes occur (i.e., erratic X-ray exposure resulting in rapid elution; too high a pump speed resulting in rapid elution; too many cells per filter resulting in a slow elution;
IN UNLABELED
0
6
MAMMALIAN
12
173
CELLS
I
18 0 6 HOURS OF ELUSION
12
18
FIG. 4. Upper panels: alkaline elution profiles of “C-labeled Ll210 cells determined by fluorescence (0) or by 14Cradioactivity (0). DNA interstrand crosslinking was measured in cells exposed to 0.5 PM HN2 for 30 min (right panel). All cells were irradiated with 300-R X irradiation (see Materials and Methods). The W-labeled 300-R control line (0) from the upper left panel is redrawn as a dashed line (---) in the upper right panel for reference. Lower panels: alkaline elution profiles of 1.5 x lo6 unlabeled drug-treated cells as determined by fluorescence (0) using lo4 W-labeled untreated cells as an internal standard (0).
incomplete lysis or deproteinization resulting in a slow elution) we feel that it is advantageous to have an internal monitor for the elution anomalies described above. To this end we have used a radioactive internal standard in the fluorometric assay. When l-2 x 106unlabeled cells are being analyzed, we have included lo4 14C-labeled cells as an internal standard. These cells are mixed with the test cells following the DNA damaging insult, and prior to X irradiation, when used. These cells then provide a reference DNA and a monitor of anomalies. (See Ref. (3) for calculations using an internal standard.) Since the 14C cells contain less than 1% of the total DNA. their
174
ERICKSON
fluorometric contribution is negligible. Following fluorometric analysis, liquid scintillation cocktail is added, and the elution pattern on the 14C-labeled cells is determined. When the two sets of data are combined, the resulting graphs give a measure of druginduced DNA crosslinking in the unlabeled cells when compared to the 14Creference cell line. Comparison of the upper and lower panels of Fig. 4 shows that the data are virtually identical. Had there been an elution anomaly in the experiment shown in the lower panels it would have been detectable in the [14C]DNA. On the contrary, had an anomaly occurred in the upper panels (i.e., a “fast” or “slow” elution) it would have gone undetected for lack of an internal standard. In this report we have described a fluorometric alkaline elution procedure which can be used for the. measurement of a variety of DNA lesions in unlabeled cells. We have tested each of the steps of this procedure, and have emphasized the necessity for a reference DNA in the fluorometric analysis. Using these procedures it should be possible to measure any of the DNA lesions currently analyzed by alkaline elution of DNA from radioactively labeled cells. We are currently using this procedure to measure the DNA damage produced in unlabeled cells isolated from tissues exposed to anti-tumor agents in vivo . ACKNOWLEDGMENTS The authors wish to thank Prof. Dr. C. G. Schmidt, Director of the Innere Klinik (Tumorforschung), Universitaetsklinikum Essen, Federal Republic of Germany, for providing the opportunity for L.C.E. to visit and work at the Universitaetsklinik, January 1979. Thanks are also given to Mr. Michael Young, Millipore Corporation, for assisting in the application of Durapore filters to this procedure. We wish to thank Mrs. Irene Clark for expert assistance with cell cultures, and Ms. Madie Tyler for the preparation of the manuscript.
REFERENCES 1. Kohn, K. W., and Ewig, R. A, G. (1973) Cancer Res. 33, 1849-1853. 2. Kohn, K. W., Erickson, R. A. G., and Friedman, C. A. (1976) Biochemistry 15, 4629-4637. 3. Kohn, K. W., Ewig, R. A. G., Erickson, L. C.,
ET AL.
4. 5. 6. 7. 8. 9. 10. 11.
and Zwelling, L. A. (1980) in Handbook of DNA Repair Techniques (Friedberg, E., and HanaWalt, P. eds.), Dekker, New York, in press. Fomace, A. J., Kohn, K. W., and Kann, H. E. (1976) Proc. Nat. Acad. Sci. USA 73, 39-43. Iqbal, Z. M., Kohn, K. W., and Ewig, R. A. G. (1976) Cancer Res. 36, 3834-3838. Swenberg, J. A., Petzold, G. L., and Harbach, P. R. (1976) Biochem. Biophys. Res. Commun. 72(6), 732-738. Bradley, M. O., Erickson, L. C., and Kohn, K. W. (1976) Mutat. Res. 37, 279-292. Erickson, L. C., Bradley, M. O., and Kohn, K. W. (1977) Cancer Res. 37, 3744-3750. Bradley, M. O., Erickson, L. C., and Kohn, K. W. (1978) Biochim. Biophys. Acta 520, 11-20. Petzold, G. L., and Swenberg, J. A. (1978) Cancer Res. 38, 1589-1594. Eastman, A., and Bresnick, E. (1978)Chem.-Biol. Interac.
23, 369-377.
12. Erickson, L. C., Bradley, M. O., and Kohn, K. W. (1978) Cancer Res. 38, 3379-3384. 13. Parodi, S., Taninagher, M., Santi, L., Cavanna, M., Sciaba, L., Maura, A., and Brambilla, G. (1978) Mutat. Res. 54, 39-46. 14. Fomace, A. J., and Kohn, K. W. (1976) Biochim. Biophys. Acta 435, 95- 103. 15. Fornace, A. J., and Little, J. B. (1977) Biochim. Biophys.
Acta
477, 343-355.
16. Ewig, R. A. G., and Kohn, K. W. (1978) Res.
Cancer
38, 3197-3203.
17. Zwelling, L. A., Anderson, T., and Kohn, K. W. (1979) Cartcer Res. 39, 365-369. 18. Kohn, K. W., and Ewig, R. A. G. (1979) Biochim. Biophys.
Acta
562,
32-40.
19. Erickson, L. C., Bradley, M. O., Ducore, J. M., Ewig, R. A. G., and Kohn, K. W. (1980) Proc. Nat. Acad. Sci. USA 77(l), 467-471. 20. Ewig, R. A. G., and Kohn, K. W. (1977) Cancer Res. 37, 2114-2122. 21. Erickson, L. C., Osieka, R., and Kohn, K. W. (1978) Cancer Res. 38, 802-808. 22. Ross, W. E., Ewig, R. A. G., and Kohn, K. W. (1978) Cancer Res. 38, 1502- 1506. 23. Zwelling, L. A., Kohn, K. W., Ross, W. E., Ewig, R. A. G., and Anderson, T. (1978) Cancer Res. 38, 1762- 1768. 24. Thomas, C. B., Osieka, R., and Kohn, K. W. (1978) Cancer Res. 38,2448-2454. 25. Ross, W. E., Glaubiger, D. L., and Kohn, K. W. (1978) Biochim. Biophys. Acta 519, 23-30. 26. Ross, W. E., Glaubiger, D. L., and Kohn, K. W. (1979) Biochim. Biophys. Acta 562, 41-5’0. 27. Bradley, M. 0.. and Kohn, K. W. (1979) Nucleic Acid.
Res.
7, 793-804.
28. Erickson, L. C., Ross, W. E., and Kohn, K. W. (1979) Chromosomea 74, 125- 139. 29. Kissane, J. M., and Robbins, E. (1958) J. Biol. Chem.
233,
184-188.
BIOCHEMISTRY
106,169-174
(1980)
Measurement of DNA Damage in Unlabeled Mammalian Cells Analyzed by Alkaline Elution and a Fluorometric DNA Assay LEONARD C. ERICKSON,‘RAINHARDT OSIEKA,~ NANCY A. SHARKEY, ANDKURT W. KOHN Laboratory
of Molecular Pharmacology, National Cancer Institute,
Developmental Therapeutics National Institutes of Healih,
Program, Bethesda,
Division of Cancer Maryland 20205
Treatment,
Received January 25, 1980 A fluorometric procedure is described that can be used in the alkaline elution technique for the measurement of DNA damage in cells whose DNA is not, or cannot be, radioactively labeled. The procedure can be used for the measurement of DNA single-strand breaks, DNA-protein crosslinking, and DNA interstrand crosslinking, and possibly other DNA lesions produced in unlabeled cells. Although developed for the measurement of DNA damage in tissue-cultured cells, the technique is applicable to the measurement of DNA damage in cells isolated from tissues exposed to DNA damaging agents in vivo.
The alkaline elution technique, developed in this laboratory (1 - 3), has proven to be a powerful and sensitive method for the measurement of a variety of DNA lesions produced in mammalian cells by various types of DNA-damaging agents. The alkaline elution method measures damage in the DNA of cells which have been immobilized on a membrane filter, and lysed with detergent. Following lysis, an alkaline solution of pH 12.1 is slowly pumped through the filter. DNA is eluted from the filter as a function of molecular weight, with lowmolecular-weight DNA eluting rapidly, and high-molecular-weight DNA eluting at later times. Among the lesions measured by alkaline elution are DNA single-strand breaks (4- 13), DNA-protein crosslinks (14- 19), DNA interstrand crosslinks (16,17,19,20-24), protein-concealed single-strand breaks (25,26), and DNA double-strand breaks (27). The alkaline elution technique has 1 To whom requests for reprints should be addressed at Building 37, Room 5D17, National Institutes of Health, Bethesda, Md. 20205. * The Innere Klinik (Tumorforschung) Universitaetsklinikum Essen, Federal Republic of Germany. 169
also proven useful for the isolation of DNA replication intermediates (28). In all of the referenced studies it was necessary to prelabel the cellular DNA with [‘“Cl- or [3H]thymidine prior to alkaline elution analysis. Recently several laboratories, including our own, have attempted to measure DNA singlestrand breaks by fluorometric analysis of eluted DNA (9,13). However, no fully documented procedure has been published, testing the completeness of DNA recoveries and the validity of the assays. We now report a convenient and accurate fluorometric procedure applicable to unlabeled cells and tissues analyzed by the alkaline elution technique. MATERIALS
AND METHODS
Cell culture. L1210 mouse leukemia cells were maintained in suspension culture in RPM1 1630 medium, supplemented with 20% heat-inactivated fetal calf serum (Flow Laboratories, Rockville, Md.), penicillin, and streptomycin. Cultures were maintained in exponential growth phase with a doubling time of about 12 h. Cells in experiments 0003-2697/80/l 10169~06$02.00/0 Copyright All rights
0 1980 by Academic Rcss, Inc. of reproduction in any form reserved.
170
ERICKSON
were used either unlabeled, or the DNA was labeled with [2-14C]thymidine (>50 mCi/mM, New England Nuclear, Boston, Mass.). Labeled cells were grown for 20 h in either 0.02 pCi/ml [14C]thymidine, or 0.2 pCi/ml [14C]thymidine as indicated in the text. HN23 was obtained from the Drug Development Branch, National Cancer Institute, NIH, Bethesda, Maryland. The drug was dissolved in 0.1 N HCl and stock solutions were frozen at -20°C. The drug was added to cultures of L1210 cells at a concentration of 1.0 x IO6 cells/ml in 1630 medium containing 20% fetal calf serum. Drug exposures were terminated by two centrifugations at SOOg for 5 min and resuspension in fresh 1630 medium. Alkaline elution. The alkaline elution procedure used has been described in detail in several recent publications (2,3). For the present experiments following drug treatment, cells were chilled on ice in 1630 medium, irradiated with 300-R X rays, and analyzed by alkaline elution. X irradiation was delivered by two Philips RT-250 X-ray tubes operating at 200 KeV and 15 mA, with 0.25-mm copper and 0.55~mm aluminum filters. Following irradiation, the cells were resuspended in 15 ml ice-cold PBS, and deposited by mild suction on a 2.0-km-poresize polyvinyl chloride (PVC) filter (Millipore Type BSWP 02500, Millipore Corp., Bedford, Mass.). The cells were immediately lysed with 5 ml of a solution of 2% sodium lauryl sulfate (SDS), 0.02 M EDTA, 0.1 M glycine (pH 10.0) which was allowed to drip through the filter by gravity. The lysate was deproteinized by pumping lysing solution containing 0.5 mg/ml proteinase-K (E. M. Laboratory, Elmsford N. Y.) through the filter at 0.035 ml/min for 30 min. The remaining proteinase solution was gently 3 Abbreviations used: HN2, bis(2-chloroethyl)methylamine; PBS, phosphate-buffered saline, 0.15 M NaW0.014 M KH,POd0.086 M K,HPOI; DABA, 35 diaminobenzoic acid, SDS, sodium lauryl sulfate.
ET AL.
aspirated off, and a solution containing 0.02 M EDTA, 0.1% SDS, and tetrapropylammonium hydroxide to yield a pH of 12.1, was added and pumped through the filter at 0.035 mYmin. Five 3-h (6.3-m]) fractions were collected into scintillation vials. At the end of the elution, the remaining solution was decanted off the filter, and the filter removed to a clean scintillation vial and air dried. A 5-ml wash of tetrapropylammonium hydroxide was pumped through the filter holder and pump lines, and was collected and processed as the sixth fraction. Sample processing and fluorescence measurement. In the standard alkaline elution assay, since the DNA is radioactively labeled, samples are processed by the addition of a liquid scintillation cocktail to the eluted fractions, and counting in a liquid scintillation counter. For the fluorometric analysis, DNA in the eluted fractions must be quantitatively and reproducibly recovered from the eluting solution. DNA was recovered by the addition of 2 vol of 95% ethanol containing 0.3 M sodium acetate (high purity, high-pressure liquid chromatography grade, Fisher Scientific, Pittsburgh, Pa.), and 0.055 M glacial acetic acid. The samples were capped, mixed, and chilled to -20°C for 30 min. The precipitated DNA in each fraction was trapped on a 0.2+mpore-size Durapore filter (type GVWP 02500, Millipore Corp.) using a 30-position Millipore sampling manifold. Vials and filters were washed with a 50% ethanol solution, the filters removed to clean scintillation vials, and air dried. Following air drying, all filters were processed by a modification of the fluorometric technique of Kissane and Robbins (29). A 2 M DABA solution (3,5-diaminobenzoic acid dihydrochloride, Aldrich Chemicals, Milwaukee, Wise.) was prepared with glass-distilled water, shaken with 50 mg/ml activated charcoal (Sigma, St. Louis, MO.), and filtered through a 0.2~pm-poresize Millipore Sterifil D-GS filtration unit.
MEASUREMENT
OF DNA DAMAGE
DABA solution, 400 ~1, was added to each filter (eluted fraction precipitate, and PVC elution filter), the vials were tightly capped with polyethylene-lined caps, and heated at 60°C for 45 min. Following heating, 1.6 ml 1 N HCl was added to each vial, the vials were shaken vigorously, and the fluorescence was determined. Three Durapore and three PVC blank filters were prepared for each experiment by running the appropriate solutions through them. This was necessary to accurately establish the background fluorescence for each filter. Additionally, a standard curve of precipitated DNA (O-10 pg) was routinely prepared for each experiment. Fluorescence was read in a Farrand Ratio-2 fluorometer (FOCI Corp., Valhalla, N. Y .) equipped with two 405nm interference filters in series for excitation wavelengths, and two 525nm interference filters for emission readout. Samples were pumped into a flow cuvette using a peristaltic pump, and recovered for scintillation counting by reversal of the peristaltic pump. In this way fluorescence and radioactivity could be determined in the same sample. For liquid scintillation counting, 10 ml Aquassure Liquid scintillation cocktail (New England Nuclear) was added to each sample, and the samples were counted in a Packard 2450B liquid scintillation counter. The DABA/ Aquassure cocktail is highly quenched, however, carbon-14 can be conveniently counted using an open tritium window at an efficiency of approximately 50%. RESULTS AND DISCUSSION
To accurately measure DNA damage by alkaline elution, DNA must be quantitatively and reproducibly recovered from the eluted fractions and the elution filters. The data presented in this study demonstrate that these criteria can be met using the procedure as described. After testing many types of filters which were unacceptable in their DNA trapping ability, wetability, or compatability with
IN UNLABELED
MAMMALIAN
CELLS
171
CPM IN 1 ml ALIQUOT OF DABA SOLUTION lx 10’1
FIG. 1, Extraction of precipitated W-labeled DNA from Durapore filters (see Materials and Methods). Following incubation with DABA and addition of HCl as described under Materials and Methods (total volume 2 ml), 1 ml of DABA solution was removed, a blank Durapore filter added, and the r4C counts per minute were determined in a liquid scintillation counter. The counts per minute observed are plotted versus the counts per minute in the remaining 1 ml of DABA solution containing the original precipitation filter. Equivalence is indicated by the dashed diagonal line. Three replicate determinations are plotted for the following DNA concentrations: 2.5,5,7.5, and 1Opgof added DNA.
DABA, we chose O.Zpm-pore-size Durapore filters (Millipore, type GVWP) to recover DNA in the eluted fractions. Studies with radioactive DNA demonstrated a trapping efficiency of better than 98% for DNA precipitated out of pH 12.1 tetrapropylammonium hydroxide, when mixed with 2 vol of ethanol/sodium acetate as described under Materials and Methods. When known amounts of unlabeled calf thymus DNA were precipitated onto Durapore filters from the eluting solution, reacted with DABA, and read in a fluorometer, a highly reproducible linear response to DNA concentration was observed. A DNA concentration of 10 pg/sample yielded fluorometric readings of approximately 0.30 in our system. Although the trapping efficiency of the filter is very important in the recovery of the DNA from the eluted fractions, it is also necessary to show that the precipitated DNA can be accurately recovered from the
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t w 0.30 1 0.20 s2 0.10 0 0.00
l?Ll
2.5 CELLS
7.5 5.0 PER FILTER (x 10’)
10.0
FIG. 2. Determination of the amount of DNA retained on the 2.0-pm-pore-size PVC filter (Millipore, type BSWP) elution filter. The indicated number of “Clabeled L1210 cells was added to the filters, lysed as described under Materials and Methods, and tetrapropylammonium hydroxide (pH 12.1) was pumped through the filter for 1 h. The filters were removed from the funnels, air dried, reacted with DABA as described, and the fluorescence at 525 nm determined. Symbols indicate the mean k 1 SD for three replicate determinations.
precipitation filter. Figure 1 shows that DNA precipitated onto Durapore filters can be completely extracted from the filter by heating in 2 M DABA. A small quantity of high-specific-activity 14C-labeled L1210 DNA was added to unlabeled calf thymus DNA, precipitated, heated in 400 ~1 of DABA, and 1.6 ml 1 N HCI was added. The solution and filter were shaken vigorously and 1 ml of DABA/HCl solution was removed and added to a scintillation vial containing a blank Durapore filter. Ten milliliters of Aquassure LSC fluid were added to this vial and to the original vial with the precipitation filter plus the remaining 1 ml of DABA/HCl, and both vials were counted in a liquid scintillation counter. This test was performed in triplicate, with four concentrations of added DNA: 2.5, 5, 7.5, and 10 pg DNA. If the DNA had not been completely extracted from the Durapore filter, more of the 14C counts per minute should have been observed in the 1 ml DABA/HCl plus the original Durapore precipitation filter. Figure 1 shows that this was not the case. It can be seen that the
ET AL.
counts per minute in both samples of DAB& HCI are equivalent, indicating that recovery of DNA from the filter is complete. Equally as important is the quantitative and precise recovery of the DNA remaining on the PVC filter at the end of the elution. Figure 2 shows that the fluorescence observed following treatment of the PVC filter with DABA is in fact reproducible and proportional to the cell number added to the filter. This point is extremely critical, since the DNA remaining on the filter is a critical value in the assessment of DNA damage in treated cells. Figure 3 shows that, as was demonstrated from the Durapore filter in Fig. 1, the extraction of the DNA from the PVC filter is complete. These data were obtained by measuring the radioactivity remaining on the filter from 2.5, 5, 7.5, and 10 x lo6 14C-labeled L1210 cells added to the filter as described in the legends of Figs. 2 and 3. When the fluorescence obtained with ulfiltered DNA (air dried in a vial) was compared to the fluorescence of the same quantity of DNA precipitated onto a filter, we found approximately 50% quenching of the readings. This may be due to the variety of reagents run through the filters or the filter material
7.5 10.0 2.5 5.0 CPM IN 1 ml ALIQUOT OF DABA SOLUTION (x 10’) FIG. 3. Extraction of “C-labeled DNA from the PVC filters shown in Fig. 2. The protocol used was identical to that described in Fig. 1. Individual symbols indicate three replicate determinations for 2.5, 5, 7.5, and 10 x 10s cells used per filter. Diagonal dashed line indicates equivalence.
MEASUREMENT
OF DNA DAMAGE
itself. When the Durapore and PVC filters were compared for quench characteristics by air drying a DNA solution onto mock filters, the quenching of fluorescence by the two filters was virtually identical. Therefore, although some quenching of fluorescence occurs from the procedure, it is similar in both filter types so that no correction factor between the filters is necessary. For a fluorometric application of the alkaline elution technique to be useful it must give results qualitatively and quantitatively similar to the standard radioactive alkaline elution procedure. This is shown to be the case in Fig. 4, where the interstrand crosslinking produced by the anti-tumor agent HN2 (20,22) has been measured by fluorescence, and radioactivity in the same cells (upper panels). In this experiment 1.5 x lo6 14C-labeled cells were analyzed and the elution pattern determined by the fluorometric procedure is shown by solid symbols, and the elution pattern determined by radioactivity is shown by open symbols. To measure DNA interstrand crosslinking, the cells are irradiated with 300-R X rays. Interstrand crosslinking reduces the elution rate of DNA containing crosslinks. For this reason the 300 R only control pattern of the upper left panel is reproduced in the right panel for reference. These results demonstrate that the fluorometric analysis is in close aggreement with the radioactive analysis. Similar results were obtained with the anti-tumor agents cis-platinum(I1) diaminedichloride (23), and L-phenylalanine mustard (22), data not shown. The lower panels of Fig. 4 illustrate fluorometric elution assays of drug-treated unlabeled cells in which quantitation of interstrand crosslinking has been improved by the addition of a small number of untreated 14C-labeled cells as an internal reference. Because elution problems sometimes occur (i.e., erratic X-ray exposure resulting in rapid elution; too high a pump speed resulting in rapid elution; too many cells per filter resulting in a slow elution;
IN UNLABELED
0
6
MAMMALIAN
12
173
CELLS
I
18 0 6 HOURS OF ELUSION
12
18
FIG. 4. Upper panels: alkaline elution profiles of “C-labeled Ll210 cells determined by fluorescence (0) or by 14Cradioactivity (0). DNA interstrand crosslinking was measured in cells exposed to 0.5 PM HN2 for 30 min (right panel). All cells were irradiated with 300-R X irradiation (see Materials and Methods). The W-labeled 300-R control line (0) from the upper left panel is redrawn as a dashed line (---) in the upper right panel for reference. Lower panels: alkaline elution profiles of 1.5 x lo6 unlabeled drug-treated cells as determined by fluorescence (0) using lo4 W-labeled untreated cells as an internal standard (0).
incomplete lysis or deproteinization resulting in a slow elution) we feel that it is advantageous to have an internal monitor for the elution anomalies described above. To this end we have used a radioactive internal standard in the fluorometric assay. When l-2 x 106unlabeled cells are being analyzed, we have included lo4 14C-labeled cells as an internal standard. These cells are mixed with the test cells following the DNA damaging insult, and prior to X irradiation, when used. These cells then provide a reference DNA and a monitor of anomalies. (See Ref. (3) for calculations using an internal standard.) Since the 14C cells contain less than 1% of the total DNA. their
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fluorometric contribution is negligible. Following fluorometric analysis, liquid scintillation cocktail is added, and the elution pattern on the 14C-labeled cells is determined. When the two sets of data are combined, the resulting graphs give a measure of druginduced DNA crosslinking in the unlabeled cells when compared to the 14Creference cell line. Comparison of the upper and lower panels of Fig. 4 shows that the data are virtually identical. Had there been an elution anomaly in the experiment shown in the lower panels it would have been detectable in the [14C]DNA. On the contrary, had an anomaly occurred in the upper panels (i.e., a “fast” or “slow” elution) it would have gone undetected for lack of an internal standard. In this report we have described a fluorometric alkaline elution procedure which can be used for the. measurement of a variety of DNA lesions in unlabeled cells. We have tested each of the steps of this procedure, and have emphasized the necessity for a reference DNA in the fluorometric analysis. Using these procedures it should be possible to measure any of the DNA lesions currently analyzed by alkaline elution of DNA from radioactively labeled cells. We are currently using this procedure to measure the DNA damage produced in unlabeled cells isolated from tissues exposed to anti-tumor agents in vivo . ACKNOWLEDGMENTS The authors wish to thank Prof. Dr. C. G. Schmidt, Director of the Innere Klinik (Tumorforschung), Universitaetsklinikum Essen, Federal Republic of Germany, for providing the opportunity for L.C.E. to visit and work at the Universitaetsklinik, January 1979. Thanks are also given to Mr. Michael Young, Millipore Corporation, for assisting in the application of Durapore filters to this procedure. We wish to thank Mrs. Irene Clark for expert assistance with cell cultures, and Ms. Madie Tyler for the preparation of the manuscript.
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ET AL.
4. 5. 6. 7. 8. 9. 10. 11.
and Zwelling, L. A. (1980) in Handbook of DNA Repair Techniques (Friedberg, E., and HanaWalt, P. eds.), Dekker, New York, in press. Fomace, A. J., Kohn, K. W., and Kann, H. E. (1976) Proc. Nat. Acad. Sci. USA 73, 39-43. Iqbal, Z. M., Kohn, K. W., and Ewig, R. A. G. (1976) Cancer Res. 36, 3834-3838. Swenberg, J. A., Petzold, G. L., and Harbach, P. R. (1976) Biochem. Biophys. Res. Commun. 72(6), 732-738. Bradley, M. O., Erickson, L. C., and Kohn, K. W. (1976) Mutat. Res. 37, 279-292. Erickson, L. C., Bradley, M. O., and Kohn, K. W. (1977) Cancer Res. 37, 3744-3750. Bradley, M. O., Erickson, L. C., and Kohn, K. W. (1978) Biochim. Biophys. Acta 520, 11-20. Petzold, G. L., and Swenberg, J. A. (1978) Cancer Res. 38, 1589-1594. Eastman, A., and Bresnick, E. (1978)Chem.-Biol. Interac.
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