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Immunocytological detection of AAF-DNA adducts in HeLa cell nuclei
193
Cancer Letters, 14 (1981) 193-204 Elsevier/North-Holland Scientific Publishers Ltd.
JMMUNOCYTOLOGICAL HELA CELL NUCLEI
DETECTION OF AAF-DNA
EVELYNE SAGEa, NORMAN GABELMANb, ROBERT BASESbqi
ADDUCTS IN
FRANCES MENDEZb and
aCentre de Biophysique Moleculaire, CNRS lA, Avenue de la Recherche Scientifique, 45045 Orleans-Cedex (France) and bDepartment of Radiology, Albert Einstein College of Medicine, Bronx, New York 10461 (U.S.A.) (Received 3 July 1981) (Revised version received 29 July 1981) (Accepted 30 July 1981)
SUMMARY
Acetylaminofluorene-DNA adducts (AAF-DNA) were detected in the nuclei of HeLa cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcAAF), using an immunocytological technique and specific antibodies directed against AAF modified DNA. The proportion of cells exhibiting specific nuclear immunoreactivity was dosedependent. The time course of disappearance of adduct specific nuclear immunoreactivity was compared with removal of N-(deoxyguanosin-&yl)-2-acetylaminofluorene (dG-C&AAF) and other adducts.
INTRODUCTION
Studies on the fate of specific carcinogen adducts in the nucleic acids of exposed cells have usually focused on biochemical and radioimmune assays [1,2,6,17,25,26]. Experiments described here provide, for the first time, an immunocytological means to study the appearance of adduct antigens in intact cells. Antibodies directed against nucleosides are base specific and react exclusively with single-stranded nucleic acids [lo]. A single specific base exposed in a segment of single-stranded DNA only 4---5 bases in length can be detected [ 301. We previously studied DNA unwinding after DNA damage by detecting exposed normal bases in animal cell DNA by immunocytological techniques [4,5,23]. Base specificity was verified by hapten inhibition [lo], in vitro, and in immunocytological tests (unpublished *Author to whom correspondence 0304-3835/81/0000-0000/$02.50 o 1981 Elsevier/North-Holland
should be addressed.
Scientific Publishers Ltd.
194
observations). Here we present studies on rabbit antibodies directed against AAF-guanosine and antibodies against DNA which had been modified by the presence of AAF adducts [21,29]. These antibodies are specific for the adduct attached to the C-8 of guanosine [9,25]. MATERIALS
AND METHODS
Cell culture methods and fluorescent antibody staining procedures have been described in previous publications [4,5,7,8,23,24]. N-AC-AAF was obtained from Midwest Research Institute, Kansas City, MO 64110 through the courtesy of Dr. D. G. Longfellow, Cancer Cause and Prevention, NCI. In certain experiments [ 3H]N-acetoxy-2-acetylaminofluorene, 3 50 mCi/ mmol, uniformly labeled, was used. Antibodies against DNA modified by N-AC-AAF were purified as previously described [ 211. Antibodies directed against AAF-guanosine were raised in rabbits using the bovine serum albumin (BSA) conjugate of the carcinogen-modified guanosine. The antibodies were purified by absorption with BSA and their specificity was verified by gel diffusion. Cells were fixed on slides and stained for 30 min with anti-AAF-DNA at a concentration of 0.24 mg/ml. For anti-AAF-guanosine, the antibody concentration was 0.14 mg/ml. It was necessary to pretreat the cells with RNases before staining with these antibodies, because of cross-reactions with nuclear RNA [4,5]. Interference from RNA is completely eliminated by pretreating fixed cells with 2 units/ml of T-l and 2 pg/ml of pancreatic ribonuclease at room temperature for 15 min [ 71. After the first layer of antibody the slides were rinsed with phosphate buffered saline, stained with fluorescein isothiocyanate (FITC) labeled sheep antirabbit immunoglobulin (Institut Pasteur No. 01-7456) at a concentration of 0.20 mg/ml for 15 min and examined with a Zeiss U.V. microscope. Two different antibodies were used as first layers. The one prepared in Orleans (E.S.) was specific for AAF-modified DNA; the one prepared here was specific for AAF-guanosine. FITC labeled sheep anti-rabbit immunoglobulin served as a common second layer, permitting direct comparison of the different first layers (FITC labeled goat anti-rabbit immunoglobulin was also used). The nuclei of fixed stained cells accounted for most of the cross-sectional areas examined. Cytoplasmic fluorescence was ignored. We relied on detecting multiple sharp, bright, or punctate foci of intranuclear detail for scoring. The nuclear membrane is prominent in positively stained cells. By changing the depth of focus for every cell examined, an observer can discriminate between the specific detail described above and the lumpy or homogeneous brightness sometimes encountered in the overlying cytoplasm. Brightness alone cannot be relied upon. Each value shown in the figures represents results of determinations independently made by 2 observers (E.S. and R.B.) in 2 or more staining
195
sessions. For each point shown, at least 800 cells were examined; results obtained by the 2 observers were not significantly different. RESULTS
AND DISCUSSION
HeLa cells growing in suspension at 2.5 X lO’/ml were exposed to increasing concentrations of N-AC-AAF in dimethylsulfoxide at less than 0.5% final concentration. Figure 2 shows a dose-dependent increase in the proportion of immunoreactive cells in cultures exposed to the carcinogen for 15 min at 37°C. The background of lo-15% may be accounted for by a contribution to intranuclear staining from naturally occurring antibodies to DNA, which can be found in certain normal sera [28]. Previous studies had shown that the antibody preparations used in the experiment of Figs. 2 and 3 with anti AAF-DNA, and Fig. 4 with anti AAF-guanosine, were highly specific for the adduct [13,21,29 and unpublished observations]. We anticipated detecting adducts in all exposed cells after exposure to these high levels of N-AC-AAF became autoradiographic studies have indicated that 99% of cells have AAF bound to DNA at such doses, but the specific staining in the experiment of Fig. 2 showed only 40% of the nuclei could be scored as positive. To facilitate scoring positive cells, the concentration of antibody was increased from 0.06 mg/ml of specific anti AAF-DNA IgG to 0.24 mg/ml for subsequent experiments; 65% of treated cells were now readily scored as positive (Fig. 3). With 0.43 mg/ml of specific antibody under these conditions of rapid staining virtually 100% of N-AC-AAF exposed cells were scored as positive (not shown). For routine use 0.24 mg/ml was chosen since this provided greater sensitivity in kinetic studies shown below. Figure 3 shows the influence of 5 pg/ml or 1 pg/ml of the carcinogen on specific immunoreactivity in a kinetic study. With 5 gg/ml, 65% of the cells were positive. The proportion of fluorescent nuclei in the untreated cells remained 15% or less. At the time intervals shown, samples were removed from the suspension and the percent of immunoreactive nuclei was determined. The results clearly show a fall in the percent of immunoreactive cells in the first 3 h post-treatment, with return to normal levels by 21 h. Cells of the experiment of Fig. 3 were also stained with antibodies against AAF-guanosine. After its initial appearance, there again was progressive decline of carcinogen-induced immunoreactivity (Fig. 4). The slower disappearance of immunoreactivity in the experiment of Fig. 4 might be accounted for by the higher affinity of antibodies against AAF-G for the adduct [ 13,291. We previously observed induction of immunoreactivity to antinucleoside antibodies against normal bases after exposure of HeLa cells to a variety of carcinogens, including N-AC-AAF [ 51. Cells from the experiment of Fig. 3 were stained with antiguanosine antibodies at a concentration of 0.22 mg/ ml. As shown in Fig. 5, the proportion of immunoreactive cells in the
196
. ..
.
_.
_.
_-
_-
;. _ _ ,,.._
Fig. 1. HeLa cells stained after exposure to 5 rg/rnl of N-AC-AAF for 15 min. All cells were exposed to the carcinogen. Cells of photographs 1A and 1B were stained with 0.24 mg/ml normal rabbit IgG (1A) or 0.24 mg/ml of immune rabbit IgG against AAF modified DNA (IB). Both cell preparations were then stained with 0.12 mg/ml FITC labeled goat antirabbit IgG (Miles Research Product, Elkhart IN). All 11 cells of 1B were positive; only 1 cell in 1A was positive, Long arrows, positively stained cells; arrow heads, negatively stained cells. All photographs were taken at 125X magnification. Photographs 1C and 1D are of cells from the same experiment; the fist layer of IgG was 0.14 mg/ml of rabbit anti-AAF-guanosine (1D) or 0.14 mg/ml of the same rabbit’s pre-immune serum (1C). The second layer was the FITC labeled goat antirabbit IgG used above. Cells of 1D show specific staining. Photographs 1E and 1F show carcinogen exposed cells stained directly with 0.14 mg/ml of FITC labeled rabbit IgG antiguanosine (1F). As a control 0.14 mg/ ml of the same rabbit’s FITC labeled pre-immune IgG was used (1E). All cells in 1F were positive, but extreme brightness and different depths of focus obscured this in certain cells. Note the low background fluorescence when a single FITC labeled antibody layer was used (IE). A prominent fluorescent nuclear membrane helps identify specifically stained cells.
N-AC
-AAF
pg/ml
Fig. 2. Immunoreactivity to anti-AAF-DNA after exposure to N-AC-AAF in HeLa cells. Cells were exposed to the carcinogen and the percent of immunoreactive nuclei determined. l, treated cells; 0, cells treated with DNase, 170 fig/ml, in 5 mM MgCl, before antibody staining were not immunoreactive. It is necessary to include a l-s exposure to 3% ice-cold perchloric acid and saline rinses immediately before staining, to remove persistent absorbed excised immunoreactive nucleotides and oligonucleotides which otherwise interfere, Singlestrand specific S-l nuclease also abolished nuclear immuno reactivity (not shown). Standard errors of the means are shown.
70
r
01
I
I
I
21
3 HOURS
AFTER
REMOVAL
OF CARCINOGEN
Fig. 3. Time course of disappearance of immunoreactivity in N-AC-AAF treated cells. Cells were exposed to the carcinogen for 15 min at 37”C, sedimented, resuspended in growth medium and aliquots were taken for determinations at the times shown. A, cells exposed to 5 @g/ml; 0, cells exposed to 1 pg/ml; l , unexposed cells. In colony forming assays the plating efficiency was reduced to 20% of control values after exposure to 1 pg/ml and was reduced to 0.02% with 5 pg/ml. The plating efficiency of untreated HeLa cells was 40%. Anti-AAF-DNA was used (see text) (refer to Figs. 1A and 1B).
199
01
I
I
I
3 HOURS
21 AFTER
REMOVAL
OF CARCINOGEN
Fig. 4. Time course of disappearance of immunoreactivity in HeLa cell nuclei after exposure to N-AC-AAF, using immunoglobulins to AAF-G. Symbols are as in Fig. 3 (refer to Figs. 1C and ID). Cells were from the experiment of Fig. 3.
untreated population was 35%, the expected contribution of S-phase cells whose DNA normally is unwound during replication [4]. However, exposure to the carcinogen increased the percent of immunoreactive cells, indicating further unwinding of DNA in cells normally not reactive i.e., the Gl and G2 cells. The initial increase in immunoreactivity is probably due to adduct formation and base displacement [ 11,121. Specificity of antibodies to AAF-modified DNA in HeLa cell nuclei is clearly demonstrated by comparing results of the experiments of Fig. 3 and Fig. 5. The cell preparations were from a single experiment. When the N-Ac-AAF-treated cells were stained with antibodies to AAF-modified DNA, 60% of them were positive. After 3 h of further incubation in the absence of the carcinogen this proportion had rapidly declined to 30% and it returned to control values by 21 h. In marked contrast, when the cells were instead stained with antiguanosine antibodies the proportion positive was 60- --75% and was still above 50% 21 h later. This was expected, since normal nucleosides are available to single-strand specific antinucleoside antibodies during S-phase, DNA repair and G2 arrest [ 41. The carcinogen-induced immunoreactivity in the experiment of Fig. 3 was not directed toward the 4 abundant normal nucleosides but was specifically directed toward AAF-modified DNA. These results indicate the presence and subsequent removal of AAF from DNA and confirm the specificity of anti AAF-DNA. We compared the time course of disappearance of nuclear immunoreactivity toward anti AAF-DNA with the loss of 3H radiolabel in cells exposed to 5 fig/ml of 3H-labeled N-AC-AAF (Fig. 6). Once again, nuclear immunoreactivity was induced but then declined rapidly for 5 h (as in Fig. 3).
20 t
*
‘Of, 01
3 HOURS
AFTER
REMOVAL OF CARCINOGEN
Fig. 5. Induction of anti-guanosine immunoreactivity following exposure to N-AC-AAF. Symbols are as in Fig. 3. Error terms not shown were smaller than symbol size (refer to Figs. 1E and 1F). Cells were from the experiment of Fig. 3.
However, the total radiolabel in purified nuclear DNA declined much more slowly. This was observed in 2 independent labeling experiments. DNA from the 4 time points in the experiment of Fig. 6 were examined by HPLC for nucleoside adducts, through the cooperation of Dr. F.A. Beland, National Center for Toxicological Research, Jefferson, Arkansas. The dominant adduct was N-(deoxyguanosin-&yl)-2-aminofluorene(dG-CXAF), representing 58.3%, 60.1%, 62.7% and 66.7% of the total radiolabel at the 4 times studied. The single HPLC fraction which includes dG-C8-AAF and N-(guanosin-8-yl)-2-aminofluorene (G-CX-AF) accounted for 13.5%, 12.4%, 14.2% and 2.9% during the same interval. Since the contribution of G-C8-AF to this fraction is small and can be neglected, we conclude that there was a decrease in dG-CS-AAF between the third and sixth hour. This should be compared with the immunoreactivity studies of Figs. 3,4 and 6. The removal of bound carcinogen has been related to unscheduled DNA synthesis [1,32] and to recovery from potentially cytotoxic effect of carcinogen treatment [ 141. The time course of excision repair is biphasic, with an initial rapid phase lasting 3---8 h [ 17,321. Published results show relatively slow removal of labeled adducts, e.g. 40-50% of the adducts persist after 24 h in a variety of cells studied such as epithelial cells, fibroblasts, lymphocytes, animal or human cells [2,3,14,17,19,22,25,31]. In the HPLC studies above, the major fraction, dG-C8-AF, persisted in HeLa cell DNA at constant levels for the first 6 h after exposure to the carcinogen. Nevertheless, we repeatedly observed an initial rapid decrease of carcinogen specific immunoreactivity (Figs. 3, 4 and 6).
201
HOURS
AFTER REMOVAL CARCINOGEN
OF
Fig. 6. Time course of disappearance of immunoreactivity and removal of *H-labeled AAF-DNA adducts of cells treated by “H-labeled N-AC-AAF. A, cells exposed to 5 gg/ml; a, unexposed cells, l, cpm )H-labeled adducts per fig of DNA from cells exposed to 5 ~g/ml of the carcinogen. The percent of immunoreactive cell nuclei represent mean values obtained by 2 observers. Anti-AAF-DNA was used. For this experiment 8 x 10’ HeLa cells in 150 ml of growth medium were exposed to 750 rg of ‘H-labeled N-AC-AAF for 20 min at 37”C.The cells were then sedimented and resuspended in 150 ml of fresh medium; at the times shown aliquots were removed for immunocytologic staining and for determination of radiolabel in purified DNA. DNA was isolated from isolated nuclei and purified by phenol extraction with 0.2% sodium dodecylsulphate, a simplification of a previous method [ 211. DNA purity and concentration in the samples were estimated from the A260/A2,0, the ratios were 1.8 or greater. The samples were treated with ribonucleases under conditions similar to that used earlier with fixed cells. ‘H radiolabel in TCA precipitable DNA was determined by collecting the precipitated DNA on membrane filters and counting in a liquid scintillation spectrometer. Radiolabel determinations were made on duplicate lo-fig aliquots of the purified DNA. This degree of labeling indicates 1 adduct/104 nucleotides.
We may account for this as follows: antibodies against AAF modified DNA and against dG-CSAAF also recognize dG-C8-AF but the affinity is lo-fold greater toward the acetylated form of the adduct [ 21,291. Therefore, the acetylated form is likely to be much more important in determining nuclear immunoreactivity. Results of HPLC analyses above did indeed show a significant drop in the abundance in nuclear DNA of the acetylated form, presumably due to the excision of acetylated adducts between the third and the sixth hour after removal of the carcinogen. The abundance of the deacetylated form was relatively constant during the entire 6 h of incubation and presumably made a smaller total contribution to immunoreactivity. We cannot strictly account for the immunoreactivity results from the HPLC data. In the first 3 h after the removal of carcinogen the induced immunoreactivity is rapidly lost with no significant reduction in
202
labeled DNA or in the dG-C8-AAF fraction. In the next 3 h loss of immunoreactivity is insignificant but the drop in the acetylated C-8 adducts is quite dramatic. The radiolabel experiments suggest that the majority of adducts are still in the nucleus even after 6 h but they do not react with the antibody. Possibly DNA-AAF adducts somehow become inaccessible to antibody during repair even before their actual excision. Considering that the antibody reacts with 2 C-8 adducts and the label in purified DNA is distributed among 3 adducts, more sophisticated experiments than the ones reported would be required in order to more perfectly correlate the immunocytological and the HPLC results. Most cultured cells form about 90% of the total C-8 DNA adducts as deacetylated adducts [ 271. An interesting exception are primary rat hepatocytes, which form 80% of the C-8 adducts as acetylated adducts [ 15,271. Howard et al. observed that the acetylated adduct is removed much faster than the non-acetylated adduct in rat hepatocytes [15]. Removal of dG-C8-AAF may be an early rapid step in excision repair. N2-Guanyl-substituted adducts persist much longer in DNA than C8guanyl-substituted adducts (formed in vivo after treatment by AAF and 4’-fluoro-4’-acetylaminobiphenyl [ 201. This persistence could be an important factor in the carcinogenic process [ 16,18,20,33]. The immunocytologic methods described here seem generally applicable to studies on the appearance and fate of carcinogen adducts in cells in vitro with potential applications to animal and human tissues, as in our previous studies on fixed tissue sections [ 7,8,24]. ACKNOWLEDGEMENTS
This work was supported by ACS, PDT-29F; NIH, 5T32-CAOgO-60, and the Scientist Exchange Segment, US-France (NCI-INSERM) Cancer Program, R.B. is a Jane and Arnold Ginsburg Fellow in Radiology. REFERENCES 1 Ahmed, F.E. and Setlow, R.B. (1977) Different rate-limiting steps in excision repair of ultraviolet and N-acetoxy-2-acetylaminofluorenedamaged DNA in normal human fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 74, 1548-1552. 2 Amacher, D.E., Elliott, J.A. and Lieberman, M.W. (1977) Differences in removal of acetylaminofluorene and pyrimidine dimers from the DNA of cultured mammalian cells. Proc. Natl. Acad. Sci. U.S.A., 74,1553-1557. 3 Amacher, D.E. and Lieberman, M.W. (1977) Removal of acetylaminofluorene from the DNA of control and repair deficient human fibroblssts. Biochem. Biophys. Res. Commun., 74,285-290. 4 Bases, R., Mendez, F., Hsu, KC. and Liebeskind, D. (1975) Immunoreactivity to antinucleoside antibodies persists during G2 arrest in X-irradiated HeLa cells. Exp. Cell Res., 92, 505-509. 5 Bases, R., Mendez, F., Neubort, S., Liebeskind, D. and Hsu, K.C. (1976) Carcinogeninduced immunoreactivity to antinucleoside antibodies in HeLa cells. Exp. Cell Res., 103,175-181. 6 Cerutti, P.A. (1978) Repairable damage in DNA. In: DNA Repair Mechanisms, pp. l-14. Editors: P.C. Hanawalt, F. Friedberg and C. Fox. Academic Press, New York.
203 7 Chang, T.H., Liebeskind, D., Hsu, K.C., Elequin, F., Janis, M. and Bases, R. (1978) Labeling index in clinical specimens estimated by the antinucleoside antibody method. Cancer Res., 38,1012-1018. 8 Chang, T.H., Liebeskind, D., Hsu, K.C., Elequin, F., Janis, M. and Bases, R. (1979) Labeling index in clinical specimens estimated by the antinucleoside antibody method. Cancer Res., 39, 282 (Erratum). 9 DeMurcia, G., Lang, M.E., Freund, A., Fuchs, R.P.P., Duane, M.P., Sage, E. and Leng, M. (1979) Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in Col. El DNA by means of specific antibodies. Proc. Natl. Acad. Sci. U.S.A., 76,6076-6080. 10 Erlanger, B.F. and Beiser, S.M. (1964) Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc. Natl. Acad. Sci. U.S.A., 52, 68-74. 11 Fuchs, R.P.P. (1975) In vitro recognition of carcinogen-induced local denaturation sites in native DNA by S-l endonuclease from Aspergillus oryzae. Nature, 257, 151-152. 12 Grunberger, D. and Weinstein, I.B. (1976) The base displacement model: an explanation for the conformational and functional changes in nucleic acids modified by chemical carcinogens. In: Biology of Radiation Carcinogenesis, pp. 175-187. Editors: J.M. Yuhas, R.W. Tennant and J.D. Regan. Raven Press, New York. 13 Guigues, M. and Leng, M. (1979) Reactivity of antibodies to guanosine modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene. Nucleic Acids Res., 6, 733-755. 14 Heflich, R.H., Hazard, R.M., Lommel, L., Scribner, J.D., Maher, V.M. and McCormick, J.J. (1980) A comparison of the DNA binding, cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens. Chem.-Biol. Interact., 29, 43-56. 15 Howard, P.C., Casciano, D.A., Beland, F.A. and Shaddock, J.G. (1981) The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures. Carcinogenesis, in press. 16 Kadlubar, F.F. (1980) A transversion mutation hypothesis for chemical carcinogenesis by N*-substitution of guanine in DNA. Chem.-Biol. Interact., 31, 255-263. 17 Kaneko, M. and Cerutti, P.A. (1980) Excision of N-acetoxy-2-acetyl-aminofluoreneinduced DNA adducts from chromatin fractions of human fibroblasts. Cancer Res., 40,4313-4319. 18 Kriek, E. (1974) Carcinogenesis by aromatic amines. Biochim. Biophys. Acta, 355, 177-203. 19 Kriek, E. (1972) Persistent binding to a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo. Cancer Res., 32, 2042-2048. 20 Kriek, E. and Hengeveld, G.M. (1978) Reaction products of the carcinogen N-hydroxy-4-acetylamino-4fluorobiphenyl with DNA in liver and kidney of the rat. Chem.-Biol. Interact., 21, 179, 201. 21 Leng, M., Sage, E., Fuchs, R.P.P. and Daune, M.P. (1978) Antibodies to DNA modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene. FEBS Lett., 92, 207210. 22 Levinson, J.W., Konze-Thomas, B., Maher, V.M. and McCormick, J.J. (1979) Evidence for a common rate limiting step in the repair process of ultraviolet light (UV) and N-acetoxyacetylaminofluorene (N-AC-AAF) induced damage in the DNA of human fibroblasts. Proc. Am. Assoc. Cancer Res., 20, 105. 23 Liebeskind, D., Hsu, KC., Erlanger, B. and Bases, R. (1974) Immunoreactivity to antinucleoside antibodies in X-irradiated HeLa cells. Exp. Cell Res., 83, 399-405. 24 Liebeskind, D., Hsu, K.C., Elequin, F., Mendez, L., Ghossein, N., Janis, M. and Bases, R. (1977) Novel method for estimating the labeling index in clinical specimens with the use of immunoperoxidase-labeled antinucleoside antibodies. Cancer Res., 37,323-326.
204 25 Poirier, M.C., Dubin, M.A. and Yuspa, S.H. (1979) Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay. Cancer Res., 39,1377-1381. 26 Poirier, MC., Santella, R., Weinstein, I.B., Grunberger, D. and Yuspa, S.H. (1980) Quantitation of benzo(a)pyrene-deoxyguanosine adducts by radioimmunoassay. Cancer Res., 40, 412-416. 27 Poirier, M.C., Williams, G.M. and Yuspa, S.H. (1980) Effect of culture conditions, cell type and species of origin on the distribution of acetylated and deacetylated deoxyguanosine C-8 adducts of N-acetoxy-2-acetylaminofluorene. Mol. Pharmacol. 18,581-587. 28 Rubin, R.L. and Carr, R.I. (1979) Anti DNA activity of IgGF (ab’)2 from normal human serum. J. Immunol., 122,1604--1607. 29 Sage, E., Fuchs, R.P.P. and Leng, M. (1979) Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. Biochemistry, 18,1328--1332. 30 Schreck, R.R., Erlanger, B.F. and Miller, O.J. (1977) Binding of antinucleoside antibodies reveals different classes of DNA in the chromosomes of the kangaroo rat (Dipodomyr Ordii). Exp. Cell Res., 108, 403-411. 31 Scudiero, D., Norin, A., Karran, P. and Strauss, B. (1976) DNA excision repair deficiency of human peripheral blood lymphocytes treated with chemical carcinogens. Cancer Res., 36,1397-1430. 32 Tlsty, T.D. and Lieberman, M.W. (1978) The distribution of DNA repair synthesis in chromatin and its rearrangement following damage with N-acetoxy-2-acetylaminofluorene. Nucleic Acids Res., 5, 3261-3273. 33 Westra, J.G., Kriek, E. and Hittenhausen, H. (1976) Identification of the persistently bound form of the carcinogen N-acetyl-2eminofluorene to rat liver DNA in vivo. Chem.-Biol. Interact., 15, 149-164.
Cancer Letters, 14 (1981) 193-204 Elsevier/North-Holland Scientific Publishers Ltd.
JMMUNOCYTOLOGICAL HELA CELL NUCLEI
DETECTION OF AAF-DNA
EVELYNE SAGEa, NORMAN GABELMANb, ROBERT BASESbqi
ADDUCTS IN
FRANCES MENDEZb and
aCentre de Biophysique Moleculaire, CNRS lA, Avenue de la Recherche Scientifique, 45045 Orleans-Cedex (France) and bDepartment of Radiology, Albert Einstein College of Medicine, Bronx, New York 10461 (U.S.A.) (Received 3 July 1981) (Revised version received 29 July 1981) (Accepted 30 July 1981)
SUMMARY
Acetylaminofluorene-DNA adducts (AAF-DNA) were detected in the nuclei of HeLa cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcAAF), using an immunocytological technique and specific antibodies directed against AAF modified DNA. The proportion of cells exhibiting specific nuclear immunoreactivity was dosedependent. The time course of disappearance of adduct specific nuclear immunoreactivity was compared with removal of N-(deoxyguanosin-&yl)-2-acetylaminofluorene (dG-C&AAF) and other adducts.
INTRODUCTION
Studies on the fate of specific carcinogen adducts in the nucleic acids of exposed cells have usually focused on biochemical and radioimmune assays [1,2,6,17,25,26]. Experiments described here provide, for the first time, an immunocytological means to study the appearance of adduct antigens in intact cells. Antibodies directed against nucleosides are base specific and react exclusively with single-stranded nucleic acids [lo]. A single specific base exposed in a segment of single-stranded DNA only 4---5 bases in length can be detected [ 301. We previously studied DNA unwinding after DNA damage by detecting exposed normal bases in animal cell DNA by immunocytological techniques [4,5,23]. Base specificity was verified by hapten inhibition [lo], in vitro, and in immunocytological tests (unpublished *Author to whom correspondence 0304-3835/81/0000-0000/$02.50 o 1981 Elsevier/North-Holland
should be addressed.
Scientific Publishers Ltd.
194
observations). Here we present studies on rabbit antibodies directed against AAF-guanosine and antibodies against DNA which had been modified by the presence of AAF adducts [21,29]. These antibodies are specific for the adduct attached to the C-8 of guanosine [9,25]. MATERIALS
AND METHODS
Cell culture methods and fluorescent antibody staining procedures have been described in previous publications [4,5,7,8,23,24]. N-AC-AAF was obtained from Midwest Research Institute, Kansas City, MO 64110 through the courtesy of Dr. D. G. Longfellow, Cancer Cause and Prevention, NCI. In certain experiments [ 3H]N-acetoxy-2-acetylaminofluorene, 3 50 mCi/ mmol, uniformly labeled, was used. Antibodies against DNA modified by N-AC-AAF were purified as previously described [ 211. Antibodies directed against AAF-guanosine were raised in rabbits using the bovine serum albumin (BSA) conjugate of the carcinogen-modified guanosine. The antibodies were purified by absorption with BSA and their specificity was verified by gel diffusion. Cells were fixed on slides and stained for 30 min with anti-AAF-DNA at a concentration of 0.24 mg/ml. For anti-AAF-guanosine, the antibody concentration was 0.14 mg/ml. It was necessary to pretreat the cells with RNases before staining with these antibodies, because of cross-reactions with nuclear RNA [4,5]. Interference from RNA is completely eliminated by pretreating fixed cells with 2 units/ml of T-l and 2 pg/ml of pancreatic ribonuclease at room temperature for 15 min [ 71. After the first layer of antibody the slides were rinsed with phosphate buffered saline, stained with fluorescein isothiocyanate (FITC) labeled sheep antirabbit immunoglobulin (Institut Pasteur No. 01-7456) at a concentration of 0.20 mg/ml for 15 min and examined with a Zeiss U.V. microscope. Two different antibodies were used as first layers. The one prepared in Orleans (E.S.) was specific for AAF-modified DNA; the one prepared here was specific for AAF-guanosine. FITC labeled sheep anti-rabbit immunoglobulin served as a common second layer, permitting direct comparison of the different first layers (FITC labeled goat anti-rabbit immunoglobulin was also used). The nuclei of fixed stained cells accounted for most of the cross-sectional areas examined. Cytoplasmic fluorescence was ignored. We relied on detecting multiple sharp, bright, or punctate foci of intranuclear detail for scoring. The nuclear membrane is prominent in positively stained cells. By changing the depth of focus for every cell examined, an observer can discriminate between the specific detail described above and the lumpy or homogeneous brightness sometimes encountered in the overlying cytoplasm. Brightness alone cannot be relied upon. Each value shown in the figures represents results of determinations independently made by 2 observers (E.S. and R.B.) in 2 or more staining
195
sessions. For each point shown, at least 800 cells were examined; results obtained by the 2 observers were not significantly different. RESULTS
AND DISCUSSION
HeLa cells growing in suspension at 2.5 X lO’/ml were exposed to increasing concentrations of N-AC-AAF in dimethylsulfoxide at less than 0.5% final concentration. Figure 2 shows a dose-dependent increase in the proportion of immunoreactive cells in cultures exposed to the carcinogen for 15 min at 37°C. The background of lo-15% may be accounted for by a contribution to intranuclear staining from naturally occurring antibodies to DNA, which can be found in certain normal sera [28]. Previous studies had shown that the antibody preparations used in the experiment of Figs. 2 and 3 with anti AAF-DNA, and Fig. 4 with anti AAF-guanosine, were highly specific for the adduct [13,21,29 and unpublished observations]. We anticipated detecting adducts in all exposed cells after exposure to these high levels of N-AC-AAF became autoradiographic studies have indicated that 99% of cells have AAF bound to DNA at such doses, but the specific staining in the experiment of Fig. 2 showed only 40% of the nuclei could be scored as positive. To facilitate scoring positive cells, the concentration of antibody was increased from 0.06 mg/ml of specific anti AAF-DNA IgG to 0.24 mg/ml for subsequent experiments; 65% of treated cells were now readily scored as positive (Fig. 3). With 0.43 mg/ml of specific antibody under these conditions of rapid staining virtually 100% of N-AC-AAF exposed cells were scored as positive (not shown). For routine use 0.24 mg/ml was chosen since this provided greater sensitivity in kinetic studies shown below. Figure 3 shows the influence of 5 pg/ml or 1 pg/ml of the carcinogen on specific immunoreactivity in a kinetic study. With 5 gg/ml, 65% of the cells were positive. The proportion of fluorescent nuclei in the untreated cells remained 15% or less. At the time intervals shown, samples were removed from the suspension and the percent of immunoreactive nuclei was determined. The results clearly show a fall in the percent of immunoreactive cells in the first 3 h post-treatment, with return to normal levels by 21 h. Cells of the experiment of Fig. 3 were also stained with antibodies against AAF-guanosine. After its initial appearance, there again was progressive decline of carcinogen-induced immunoreactivity (Fig. 4). The slower disappearance of immunoreactivity in the experiment of Fig. 4 might be accounted for by the higher affinity of antibodies against AAF-G for the adduct [ 13,291. We previously observed induction of immunoreactivity to antinucleoside antibodies against normal bases after exposure of HeLa cells to a variety of carcinogens, including N-AC-AAF [ 51. Cells from the experiment of Fig. 3 were stained with antiguanosine antibodies at a concentration of 0.22 mg/ ml. As shown in Fig. 5, the proportion of immunoreactive cells in the
196
. ..
.
_.
_.
_-
_-
;. _ _ ,,.._
Fig. 1. HeLa cells stained after exposure to 5 rg/rnl of N-AC-AAF for 15 min. All cells were exposed to the carcinogen. Cells of photographs 1A and 1B were stained with 0.24 mg/ml normal rabbit IgG (1A) or 0.24 mg/ml of immune rabbit IgG against AAF modified DNA (IB). Both cell preparations were then stained with 0.12 mg/ml FITC labeled goat antirabbit IgG (Miles Research Product, Elkhart IN). All 11 cells of 1B were positive; only 1 cell in 1A was positive, Long arrows, positively stained cells; arrow heads, negatively stained cells. All photographs were taken at 125X magnification. Photographs 1C and 1D are of cells from the same experiment; the fist layer of IgG was 0.14 mg/ml of rabbit anti-AAF-guanosine (1D) or 0.14 mg/ml of the same rabbit’s pre-immune serum (1C). The second layer was the FITC labeled goat antirabbit IgG used above. Cells of 1D show specific staining. Photographs 1E and 1F show carcinogen exposed cells stained directly with 0.14 mg/ml of FITC labeled rabbit IgG antiguanosine (1F). As a control 0.14 mg/ ml of the same rabbit’s FITC labeled pre-immune IgG was used (1E). All cells in 1F were positive, but extreme brightness and different depths of focus obscured this in certain cells. Note the low background fluorescence when a single FITC labeled antibody layer was used (IE). A prominent fluorescent nuclear membrane helps identify specifically stained cells.
N-AC
-AAF
pg/ml
Fig. 2. Immunoreactivity to anti-AAF-DNA after exposure to N-AC-AAF in HeLa cells. Cells were exposed to the carcinogen and the percent of immunoreactive nuclei determined. l, treated cells; 0, cells treated with DNase, 170 fig/ml, in 5 mM MgCl, before antibody staining were not immunoreactive. It is necessary to include a l-s exposure to 3% ice-cold perchloric acid and saline rinses immediately before staining, to remove persistent absorbed excised immunoreactive nucleotides and oligonucleotides which otherwise interfere, Singlestrand specific S-l nuclease also abolished nuclear immuno reactivity (not shown). Standard errors of the means are shown.
70
r
01
I
I
I
21
3 HOURS
AFTER
REMOVAL
OF CARCINOGEN
Fig. 3. Time course of disappearance of immunoreactivity in N-AC-AAF treated cells. Cells were exposed to the carcinogen for 15 min at 37”C, sedimented, resuspended in growth medium and aliquots were taken for determinations at the times shown. A, cells exposed to 5 @g/ml; 0, cells exposed to 1 pg/ml; l , unexposed cells. In colony forming assays the plating efficiency was reduced to 20% of control values after exposure to 1 pg/ml and was reduced to 0.02% with 5 pg/ml. The plating efficiency of untreated HeLa cells was 40%. Anti-AAF-DNA was used (see text) (refer to Figs. 1A and 1B).
199
01
I
I
I
3 HOURS
21 AFTER
REMOVAL
OF CARCINOGEN
Fig. 4. Time course of disappearance of immunoreactivity in HeLa cell nuclei after exposure to N-AC-AAF, using immunoglobulins to AAF-G. Symbols are as in Fig. 3 (refer to Figs. 1C and ID). Cells were from the experiment of Fig. 3.
untreated population was 35%, the expected contribution of S-phase cells whose DNA normally is unwound during replication [4]. However, exposure to the carcinogen increased the percent of immunoreactive cells, indicating further unwinding of DNA in cells normally not reactive i.e., the Gl and G2 cells. The initial increase in immunoreactivity is probably due to adduct formation and base displacement [ 11,121. Specificity of antibodies to AAF-modified DNA in HeLa cell nuclei is clearly demonstrated by comparing results of the experiments of Fig. 3 and Fig. 5. The cell preparations were from a single experiment. When the N-Ac-AAF-treated cells were stained with antibodies to AAF-modified DNA, 60% of them were positive. After 3 h of further incubation in the absence of the carcinogen this proportion had rapidly declined to 30% and it returned to control values by 21 h. In marked contrast, when the cells were instead stained with antiguanosine antibodies the proportion positive was 60- --75% and was still above 50% 21 h later. This was expected, since normal nucleosides are available to single-strand specific antinucleoside antibodies during S-phase, DNA repair and G2 arrest [ 41. The carcinogen-induced immunoreactivity in the experiment of Fig. 3 was not directed toward the 4 abundant normal nucleosides but was specifically directed toward AAF-modified DNA. These results indicate the presence and subsequent removal of AAF from DNA and confirm the specificity of anti AAF-DNA. We compared the time course of disappearance of nuclear immunoreactivity toward anti AAF-DNA with the loss of 3H radiolabel in cells exposed to 5 fig/ml of 3H-labeled N-AC-AAF (Fig. 6). Once again, nuclear immunoreactivity was induced but then declined rapidly for 5 h (as in Fig. 3).
20 t
*
‘Of, 01
3 HOURS
AFTER
REMOVAL OF CARCINOGEN
Fig. 5. Induction of anti-guanosine immunoreactivity following exposure to N-AC-AAF. Symbols are as in Fig. 3. Error terms not shown were smaller than symbol size (refer to Figs. 1E and 1F). Cells were from the experiment of Fig. 3.
However, the total radiolabel in purified nuclear DNA declined much more slowly. This was observed in 2 independent labeling experiments. DNA from the 4 time points in the experiment of Fig. 6 were examined by HPLC for nucleoside adducts, through the cooperation of Dr. F.A. Beland, National Center for Toxicological Research, Jefferson, Arkansas. The dominant adduct was N-(deoxyguanosin-&yl)-2-aminofluorene(dG-CXAF), representing 58.3%, 60.1%, 62.7% and 66.7% of the total radiolabel at the 4 times studied. The single HPLC fraction which includes dG-C8-AAF and N-(guanosin-8-yl)-2-aminofluorene (G-CX-AF) accounted for 13.5%, 12.4%, 14.2% and 2.9% during the same interval. Since the contribution of G-C8-AF to this fraction is small and can be neglected, we conclude that there was a decrease in dG-CS-AAF between the third and sixth hour. This should be compared with the immunoreactivity studies of Figs. 3,4 and 6. The removal of bound carcinogen has been related to unscheduled DNA synthesis [1,32] and to recovery from potentially cytotoxic effect of carcinogen treatment [ 141. The time course of excision repair is biphasic, with an initial rapid phase lasting 3---8 h [ 17,321. Published results show relatively slow removal of labeled adducts, e.g. 40-50% of the adducts persist after 24 h in a variety of cells studied such as epithelial cells, fibroblasts, lymphocytes, animal or human cells [2,3,14,17,19,22,25,31]. In the HPLC studies above, the major fraction, dG-C8-AF, persisted in HeLa cell DNA at constant levels for the first 6 h after exposure to the carcinogen. Nevertheless, we repeatedly observed an initial rapid decrease of carcinogen specific immunoreactivity (Figs. 3, 4 and 6).
201
HOURS
AFTER REMOVAL CARCINOGEN
OF
Fig. 6. Time course of disappearance of immunoreactivity and removal of *H-labeled AAF-DNA adducts of cells treated by “H-labeled N-AC-AAF. A, cells exposed to 5 gg/ml; a, unexposed cells, l, cpm )H-labeled adducts per fig of DNA from cells exposed to 5 ~g/ml of the carcinogen. The percent of immunoreactive cell nuclei represent mean values obtained by 2 observers. Anti-AAF-DNA was used. For this experiment 8 x 10’ HeLa cells in 150 ml of growth medium were exposed to 750 rg of ‘H-labeled N-AC-AAF for 20 min at 37”C.The cells were then sedimented and resuspended in 150 ml of fresh medium; at the times shown aliquots were removed for immunocytologic staining and for determination of radiolabel in purified DNA. DNA was isolated from isolated nuclei and purified by phenol extraction with 0.2% sodium dodecylsulphate, a simplification of a previous method [ 211. DNA purity and concentration in the samples were estimated from the A260/A2,0, the ratios were 1.8 or greater. The samples were treated with ribonucleases under conditions similar to that used earlier with fixed cells. ‘H radiolabel in TCA precipitable DNA was determined by collecting the precipitated DNA on membrane filters and counting in a liquid scintillation spectrometer. Radiolabel determinations were made on duplicate lo-fig aliquots of the purified DNA. This degree of labeling indicates 1 adduct/104 nucleotides.
We may account for this as follows: antibodies against AAF modified DNA and against dG-CSAAF also recognize dG-C8-AF but the affinity is lo-fold greater toward the acetylated form of the adduct [ 21,291. Therefore, the acetylated form is likely to be much more important in determining nuclear immunoreactivity. Results of HPLC analyses above did indeed show a significant drop in the abundance in nuclear DNA of the acetylated form, presumably due to the excision of acetylated adducts between the third and the sixth hour after removal of the carcinogen. The abundance of the deacetylated form was relatively constant during the entire 6 h of incubation and presumably made a smaller total contribution to immunoreactivity. We cannot strictly account for the immunoreactivity results from the HPLC data. In the first 3 h after the removal of carcinogen the induced immunoreactivity is rapidly lost with no significant reduction in
202
labeled DNA or in the dG-C8-AAF fraction. In the next 3 h loss of immunoreactivity is insignificant but the drop in the acetylated C-8 adducts is quite dramatic. The radiolabel experiments suggest that the majority of adducts are still in the nucleus even after 6 h but they do not react with the antibody. Possibly DNA-AAF adducts somehow become inaccessible to antibody during repair even before their actual excision. Considering that the antibody reacts with 2 C-8 adducts and the label in purified DNA is distributed among 3 adducts, more sophisticated experiments than the ones reported would be required in order to more perfectly correlate the immunocytological and the HPLC results. Most cultured cells form about 90% of the total C-8 DNA adducts as deacetylated adducts [ 271. An interesting exception are primary rat hepatocytes, which form 80% of the C-8 adducts as acetylated adducts [ 15,271. Howard et al. observed that the acetylated adduct is removed much faster than the non-acetylated adduct in rat hepatocytes [15]. Removal of dG-C8-AAF may be an early rapid step in excision repair. N2-Guanyl-substituted adducts persist much longer in DNA than C8guanyl-substituted adducts (formed in vivo after treatment by AAF and 4’-fluoro-4’-acetylaminobiphenyl [ 201. This persistence could be an important factor in the carcinogenic process [ 16,18,20,33]. The immunocytologic methods described here seem generally applicable to studies on the appearance and fate of carcinogen adducts in cells in vitro with potential applications to animal and human tissues, as in our previous studies on fixed tissue sections [ 7,8,24]. ACKNOWLEDGEMENTS
This work was supported by ACS, PDT-29F; NIH, 5T32-CAOgO-60, and the Scientist Exchange Segment, US-France (NCI-INSERM) Cancer Program, R.B. is a Jane and Arnold Ginsburg Fellow in Radiology. REFERENCES 1 Ahmed, F.E. and Setlow, R.B. (1977) Different rate-limiting steps in excision repair of ultraviolet and N-acetoxy-2-acetylaminofluorenedamaged DNA in normal human fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 74, 1548-1552. 2 Amacher, D.E., Elliott, J.A. and Lieberman, M.W. (1977) Differences in removal of acetylaminofluorene and pyrimidine dimers from the DNA of cultured mammalian cells. Proc. Natl. Acad. Sci. U.S.A., 74,1553-1557. 3 Amacher, D.E. and Lieberman, M.W. (1977) Removal of acetylaminofluorene from the DNA of control and repair deficient human fibroblssts. Biochem. Biophys. Res. Commun., 74,285-290. 4 Bases, R., Mendez, F., Hsu, KC. and Liebeskind, D. (1975) Immunoreactivity to antinucleoside antibodies persists during G2 arrest in X-irradiated HeLa cells. Exp. Cell Res., 92, 505-509. 5 Bases, R., Mendez, F., Neubort, S., Liebeskind, D. and Hsu, K.C. (1976) Carcinogeninduced immunoreactivity to antinucleoside antibodies in HeLa cells. Exp. Cell Res., 103,175-181. 6 Cerutti, P.A. (1978) Repairable damage in DNA. In: DNA Repair Mechanisms, pp. l-14. Editors: P.C. Hanawalt, F. Friedberg and C. Fox. Academic Press, New York.
203 7 Chang, T.H., Liebeskind, D., Hsu, K.C., Elequin, F., Janis, M. and Bases, R. (1978) Labeling index in clinical specimens estimated by the antinucleoside antibody method. Cancer Res., 38,1012-1018. 8 Chang, T.H., Liebeskind, D., Hsu, K.C., Elequin, F., Janis, M. and Bases, R. (1979) Labeling index in clinical specimens estimated by the antinucleoside antibody method. Cancer Res., 39, 282 (Erratum). 9 DeMurcia, G., Lang, M.E., Freund, A., Fuchs, R.P.P., Duane, M.P., Sage, E. and Leng, M. (1979) Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in Col. El DNA by means of specific antibodies. Proc. Natl. Acad. Sci. U.S.A., 76,6076-6080. 10 Erlanger, B.F. and Beiser, S.M. (1964) Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc. Natl. Acad. Sci. U.S.A., 52, 68-74. 11 Fuchs, R.P.P. (1975) In vitro recognition of carcinogen-induced local denaturation sites in native DNA by S-l endonuclease from Aspergillus oryzae. Nature, 257, 151-152. 12 Grunberger, D. and Weinstein, I.B. (1976) The base displacement model: an explanation for the conformational and functional changes in nucleic acids modified by chemical carcinogens. In: Biology of Radiation Carcinogenesis, pp. 175-187. Editors: J.M. Yuhas, R.W. Tennant and J.D. Regan. Raven Press, New York. 13 Guigues, M. and Leng, M. (1979) Reactivity of antibodies to guanosine modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene. Nucleic Acids Res., 6, 733-755. 14 Heflich, R.H., Hazard, R.M., Lommel, L., Scribner, J.D., Maher, V.M. and McCormick, J.J. (1980) A comparison of the DNA binding, cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens. Chem.-Biol. Interact., 29, 43-56. 15 Howard, P.C., Casciano, D.A., Beland, F.A. and Shaddock, J.G. (1981) The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures. Carcinogenesis, in press. 16 Kadlubar, F.F. (1980) A transversion mutation hypothesis for chemical carcinogenesis by N*-substitution of guanine in DNA. Chem.-Biol. Interact., 31, 255-263. 17 Kaneko, M. and Cerutti, P.A. (1980) Excision of N-acetoxy-2-acetyl-aminofluoreneinduced DNA adducts from chromatin fractions of human fibroblasts. Cancer Res., 40,4313-4319. 18 Kriek, E. (1974) Carcinogenesis by aromatic amines. Biochim. Biophys. Acta, 355, 177-203. 19 Kriek, E. (1972) Persistent binding to a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo. Cancer Res., 32, 2042-2048. 20 Kriek, E. and Hengeveld, G.M. (1978) Reaction products of the carcinogen N-hydroxy-4-acetylamino-4fluorobiphenyl with DNA in liver and kidney of the rat. Chem.-Biol. Interact., 21, 179, 201. 21 Leng, M., Sage, E., Fuchs, R.P.P. and Daune, M.P. (1978) Antibodies to DNA modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene. FEBS Lett., 92, 207210. 22 Levinson, J.W., Konze-Thomas, B., Maher, V.M. and McCormick, J.J. (1979) Evidence for a common rate limiting step in the repair process of ultraviolet light (UV) and N-acetoxyacetylaminofluorene (N-AC-AAF) induced damage in the DNA of human fibroblasts. Proc. Am. Assoc. Cancer Res., 20, 105. 23 Liebeskind, D., Hsu, KC., Erlanger, B. and Bases, R. (1974) Immunoreactivity to antinucleoside antibodies in X-irradiated HeLa cells. Exp. Cell Res., 83, 399-405. 24 Liebeskind, D., Hsu, K.C., Elequin, F., Mendez, L., Ghossein, N., Janis, M. and Bases, R. (1977) Novel method for estimating the labeling index in clinical specimens with the use of immunoperoxidase-labeled antinucleoside antibodies. Cancer Res., 37,323-326.
204 25 Poirier, M.C., Dubin, M.A. and Yuspa, S.H. (1979) Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay. Cancer Res., 39,1377-1381. 26 Poirier, MC., Santella, R., Weinstein, I.B., Grunberger, D. and Yuspa, S.H. (1980) Quantitation of benzo(a)pyrene-deoxyguanosine adducts by radioimmunoassay. Cancer Res., 40, 412-416. 27 Poirier, M.C., Williams, G.M. and Yuspa, S.H. (1980) Effect of culture conditions, cell type and species of origin on the distribution of acetylated and deacetylated deoxyguanosine C-8 adducts of N-acetoxy-2-acetylaminofluorene. Mol. Pharmacol. 18,581-587. 28 Rubin, R.L. and Carr, R.I. (1979) Anti DNA activity of IgGF (ab’)2 from normal human serum. J. Immunol., 122,1604--1607. 29 Sage, E., Fuchs, R.P.P. and Leng, M. (1979) Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. Biochemistry, 18,1328--1332. 30 Schreck, R.R., Erlanger, B.F. and Miller, O.J. (1977) Binding of antinucleoside antibodies reveals different classes of DNA in the chromosomes of the kangaroo rat (Dipodomyr Ordii). Exp. Cell Res., 108, 403-411. 31 Scudiero, D., Norin, A., Karran, P. and Strauss, B. (1976) DNA excision repair deficiency of human peripheral blood lymphocytes treated with chemical carcinogens. Cancer Res., 36,1397-1430. 32 Tlsty, T.D. and Lieberman, M.W. (1978) The distribution of DNA repair synthesis in chromatin and its rearrangement following damage with N-acetoxy-2-acetylaminofluorene. Nucleic Acids Res., 5, 3261-3273. 33 Westra, J.G., Kriek, E. and Hittenhausen, H. (1976) Identification of the persistently bound form of the carcinogen N-acetyl-2eminofluorene to rat liver DNA in vivo. Chem.-Biol. Interact., 15, 149-164.