Leukemia Research Vol. 15, No. 1, pp. 9-17, 1991. Printed in Great Britain.
0145-2126/91 $3.00 + .00 Pergamon Press plc
D E T E C T I O N OF SINGLE-STRANDED DNA D A M A G E USING M O N O C L O N A L ANTI-THYMIDINE ANTIBODY AZRA RAZA,* IQBAL MEHDI,* WEN JEN Guo,* NAVEED YOUSUF,* MARGARET MASTERSON,~" SALVADOREMIRTO,:~ LAWRENCEE. MOTYKA§ and GEORGE L. MAYERS§ *Barrett Cancer Center, University of Cincinnati and tChildren's Hospital Medical Center of Cincinnati, 231 Bethesda Avenue, ML 562, Rm 6367, Cincinnati, OH 45208, U.S.A., ~:Ospedale "V. Cervello", Divisione Di Ematoiogia, Palermo, Italy, §Roswell Park Memorial Institute, Department of Molecular Immunology, 666 Elm Street, Buffalo, NY 14263, U.S.A. (Received 27 October 1989. Revision accepted 6 June 1990) Abstract--A method to detect single-stranded DNA damage from individual cells has been developed
using a monoclonal anti-thymidine antibody (MoAb20B7). Initially, HL-60 cells were incubated with daunomycin at different concentrations, and processed by MoAb20B7. While 73.5% of the cells incubated with 5 ~tg/ml of daunomycin for 24 h reacted positively with MoAb20B7, 83.5% cells at 10 ~tg/ml daunomycin dose were positive. Next, this method was combined with unscheduled DNA synthesis to simultaneously measure repair and damage from individual cells. Finally, patients with acute myeloid leukemias were studied before and 24 h after therapy with a daunomycin containing regimen. In vivo damage could be determined in a prompt fashion. Key words: DNA damage, DNA repair, monoclonal anti-thymidine antibody, AML.
INTRODUCTION THE MAINSTAYof therapy in acute myeloid leukemia (AML) is a combination of cytosine arabinoside (araC) and an anthracycline such as daunomycin [1]. An accurate prediction of response to this regimen in individual cases would substantially improve treatment results since the delivery of noxious agents with little or no benefit could be avoided. A number of in vitro assays have been shown to be useful for the S-phase specific agent araC because of its ability to become incorporated in the newly synthesized strand of DNA and exert its potentially lethal effect on the cell by inhibiting further DNA synthesis [2-4]. DNA damaging agents such as the anthracyclines present a more complicated problem, since these agents are intercalators of DNA rather than becoming incorporated into DNA. Thus, their effects on the cell can only be measured indirectly. While a number of highly Abbreviations: MoAb2OB7, monoclonal anti-thymidine antibody; AML, acute myeloid leukemia; araC, cytosine arabinoside; UDS, unscheduled DNA synthesis; FCS, fetal calf serum; PBS, phosphate buffered saline; PB, peripheral blood; BM, bone marrow aspirate; HCI, hydrochloric acid; LI, labeling index; UV, ultraviolet light; MDR, multidrug resistance. Correspondence to: Dr Azra Raza, Director, Hematologic Oncology, Barrett Cancer Center, University of Cincinnati, 231 Bethesda Avenue, Room 330, ML 508, Cincinnati, OH 45267, U.S.A.
sophisticated methods to measure DNA damage have been developed in the past, most have remained confined to a few laboratories by virtue of their complexity. In addition, these methods only provided information on the population of cells as a whole rather than the individual cell [5-9]. More recently, assays have been developed to detect DNA damage from individual cells. These assays are based on antibodies produced against damaged DNA segments [10-121 . We would like to describe the development of a relatively similar method to detect single-stranded DNA damage which by virtue of its simplicity, may become an important assay for prediction of response to anthracyclines in AML. A monoclonal antibody to thymidine (MoAb20B7) was develope d based upon the hypothesis that during the repair of singlestranded damaged DNA, thymidine bases on the opposite, non-damaged strand would be available for reaction with the antibody. Thus, while in a nondamaged cell, the thymidine antibody would only react with the incorporated bases following denaturation and unwinding of the helix, single-stranded damaged DNA would be reactive with the antibody without necessitating denaturation. Moreover, this method was combined with conventional autoradiography to detect unscheduled DNA synthesis (UDS), thereby allowing us to measure DNA damage (antithymidine antibody) and repair (UDS) simultaneously from individual cells.
A. RAZAet al.
10 MATERIALS
AND METHODS
Initial work on the detection of single-stranded D N A damage and repair was conducted on the HL-60 tissue culture cell line. The characteristics of this cell line have been described at length elsewhere [13]. Briefly, it was originally derived from a patient with acute myeloid leukemia and now grows in RPMI 1640 cell culture medium supplemented with 10% fetal calf serum (FCS) and penicillin and streptomycin. In order to simplify this section, methods will be described under the following sub-headings.
1. Preparation of monoclonal anti-thymidine antibody 20B7 Hybridomas were prepared by the fusion of spleen cells from B A L B / c mice immunized with a 5-methyluridinebovine gammaglobulin conjugate [14] and P3X63-Ag8.653 myeloma cells [15] using standard procedures [16-19]. Hybridoma 20B7 was propagated in pristane treated BALB/c mice and the monoclonal anti-thymidine antibodies were isolated from the ascitic fluid. All antibodies used in these studies were affinity purified on an AHsepharose 4B-5-methyluridine affinity column. Monoclonal anti-thymidine antibody 20B7 was characterized in radioimmunoassay and shown to be highly specific for thymidine and to have no specificity for guanosine, adenosine, cytidine and to have only minimal reactivity with uridine ( 1000fold less than thymidine).
2. Detection of single-stranded MoAb2OB7
DNA
damage using
HL-60 cells in the concentration of 1 x 106/ml were incubated with daunomycin at doses of 5 ~tg and 10 ~tg/ml for 24 h. A control group of cells with no drug was also used. At the end of 24 h of incubation, ceils were washed thrice with phosphate buffered saline (PBS) at 1000 g for 5 min each and then placed on alcian-blue coated coverslips. Alcian-blue was used to cationize glass, which allows the cells to be evenly distributed throughout the coverslip and cell loss during processing is minimized due to the firm adherence produced by the electrical charge [20]. Cells were fixed in 70% ethanol for 10 min and processed by the indirect immunoperoxidase method using MoAb20B7 as described below. After fixation, the coverslips were washed in Tris buffered saline (TBS) and placed in a jar containing 100 ml of absolute methanol and 25 ml of 3% HzO2 for 20 min at room temperature (RT). This step would remove any endogenous peroxidase from cells, which was particularly important when samples of A M L patients were being studied. The coverslips were washed again in TBS and then three coverslips per group were processed by the MoAb20B7. In the first "positive control" coverslip of each group, D N A was denatured by a 20 min treatment with 4 N HC1 at RT, while no HC1 treatment was used for the actual experimental group. Coverslips from both the control and the experimental groups were then exposed to Tris buffer containing 0.01% bovine serum albumin (TrisBSA) for l h at 37°C in 5% CO2. MoAb20B7 diluted 1 : 2000 with PBS and 0.05% nonidet 40 (NP-40) was pipetted onto each coverslip for one hour at RT. Plain PBS and NP-40 without MoAb20B7 was pipetted onto the second "negative controls", i.e. cells which were treated with
daunomycin, and processed by the whole immunoperoxidase method save for the use of the primary antibody MoAb20B7. Coverslips were rinsed in PBS/Triton 100 (0.1%) for 1 h and exposed to peroxidase conjugated rabbit anti-mouse antibody (P161 from D A K O ) diluted 1:100 with TBS/4% human serum for 30 min at RT. The coverslips were rinsed in TBS followed by a 20 min exposure to freshly prepared and filtered ethylamine carbazine (AEC) at 37°C in 5% CO2. A final rinse in cold tap water was carried out and the coverslips were mounted onto glass slides with immunomount and examined under a light microscope. Any cell in which the nucleus stained anywhere from light pink to dark brown (or red) was counted as positive. At least 500-2000 cells were examined from randomly chosen fields from each coverslip and the results were presented as the percentage of positive cells.
3. Simultaneous detection of DNA damage and repair by the "double-label" technique combining anti-thymidine antibody with unscheduled DNA synthesis HL-60 cells at a concentration of 1 x 106/ml were incubated with 10-1 M hydroxyurea (HU) (Sigma Chemicals) for 2 h at 37°C in order to arrest semi-conservative D N A synthesis by S-phase cells [8]. In the last hour of the incubation, 100 ~tg of mechlorethamine/ml was added to the suspension in order to produce D N A damage. The cells were then washed thrice with RPMI 1640 and resuspended in RPMI 1640 cell culture medium at a concentration of 5 x 106 cells/ml. Freshly prepared HU at a dose of 10- l M was added to the cells along with 20 ~tl/ml of [3H]Tdr (specific activity 60-90 Ci/mmole, ICN Radiochemicals). After 2h, the cells were washed with nonradiolabeled thymidine thrice and once with PBS and placed on alcian blue coated coverslips. The coverslips were processed by the MoAb20B7 as described in Section 2 and then coated with NTB2 nuclear track emulsion (Eastman Kodak) at 42°C. The coverslips were allowed to dry completely and stored in the dark at - 6 0 to -80°C for 10 days. Kodak D-19 developer was used to develop the coverslips which were then fixed and mounted on glass slides with immunomount. Any cell with at least five silver grains was considered as positively labeled by [3H]Tdr, and ceils which stained anywhere from light pink to dark brown or red were considered as positive for MoAb20B7.
4. Detection of in vivo DNA damage in A M L patients undergoing chemotherapy with daunomycin as part of the regimen Four patients with A M L were also studied for the application of this technique to detect D N A damage induced in vivo by daunomycin. Peripheral blood (PB) and/or bone marrow (BM) aspirate samples were drawn into glass syringes containing 2% sodium citrate and light density cells were recovered following Ficoll-Hypaque separation. Cells were obtained from individual patients prior to starting remission induction chemotherapy (time 0) and serially therafter (4, 24, 48 h) following start of daunomycin containing chemotherapy combination. Following separation on the density-gradient, the mononuclear cells were placed on alcian-blue coated coverslips and processed by the single-label methodology described in Section 2. Please note that for every sample processed, two "controls" were prepared along with the "experimental" group. The first was a "positive" control in which D N A of every cell was denatured by using hydrochloric acid (HC1) so that when
DNA damage from individualcells I00
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FIo. 1. Fine specificity of monoclonal anti-thymidine antibody 20B7. This antibody shows the highest specificity for thymidine with some reactivity with the thymidine analogues 5-bromouridine and 5-iodouridine. This antibody does not react with the natural nucleosides guanosine, adenosine and cytidine and only weakly with uridine (1000fold less than thymidine).
MoAb20B7 was used following this treatment, every cell reacted positively with the antibody (since every cell contains thymidine). A "negative" control was accomplished in two parts. Firstly, control cells were processed exactly as the experimental group except that the MoAb20B7 was left out of the procedure. Secondly, an unrelated monoclonal antibody such as anti-bromodeoxyuridine which should not react with the cells being used was added in place of MoAb20B7. RESULTS In order to maintain clarity, this section will also be subdivided into separate sections.
1. Specificity of the thymidine antibody Monoclonal antibody 20B7 is one of large number of anti-thymidine antibodies whose preparation has been described in detail elsewhere (L,E.M. Ph.D. Thesis, manuscript in preparation). The fine specificity of MoAb20BT, which was isolated by affinity chromatography, has been evaluated in an R I A using thymidine, guanosine, adenosine, cytidine, uridine, 5-bromouridine, 5-iodouridine, and 5-fluorouridine as inhibitors. The results are shown in Fig. 1 which shows that this antibody does not have any specificity for guanosine, adenosine, or cytidine, and only weakly binds uridine (1000-fold weaker than thymidine). The antibody does show some weak affinity for 5-bromouridine and 5-iodouridine, the two thymidine analogues that can be incorporated into DNA in place of thymidine. In addition, in the immunocytochemical assays the binding of 20B7 can be completely inhibited by the addition of 100 ~tl of 1 x 10 - 4 M thymidine, but not by lO0~tl of 1 x 10 -v M thymidine.
11
2. Detection of single-stranded DNA damage produced by daunomycin HL-60 cells which were incubated with daunomycin at concentrations varying from 5 to 10 ~g/ml for 24 h and then processed by MoAb20B7 are shown in Figs 2 (a-c). Figure 2a represents the "negative" control where HL-60 cells were processed by the entire indirect immunoperoxidase method, except for the deletion of MoAb20B7, so that every cell is "white" or non-reactive. Similar results were obtained from the other two "negative" control groups where no HCI and no daunomycin were used, but the entire immunoperoxidase method using MoAb20B7 was performed or where anti-bromodeoxyuridine was used instead of MoAb20B7. Figure 2b is the "positive" control in which D N A was denatured by treatment with 4 N HC1 so that every cell is strongly reactive with MoAb20B7, demonstrating deep-red nuclear staining with peroxidase. Figure lc is the actual "experimental" group in which cells incubated with 5 ~tg/ml of daunomycin for 24 h were processed by MoAb20B7 and varying degrees of reactivity was noted in individual cells. Table 1 summarizes the single label data and is an average of two separate experiments. Column 1 describes the negative and positive controls first, which were noted to be universally negative and positive, for each experimental group, respectively. It can be seen that in the actual experimental group, there was an increasing percentage of cells positively reactive with MoAb20B7 in both the concentrations of daunomycin used. In the 5 ~tg/ml group, 26.5% cells were negative, 38% were 1+ reactive while 33% cells were brightly positive or 2+ reactive. In addition, 2.5% cells in this group were clearly karyorrhectic and their nuclei were noted to be undergoing fragmentation. Thus, in this group, a total of 73.5% cells were found to be reactive with the MoAb20B7. In the 10 ~tg/ml group, 16.5% cells were completely negative, 18.5% were 1 + positive, 43.5% were 2+ positive and 21.5% were karyorrhectic, giving a total of 83.5% cells reactive with the monoclonal antibody.
3. Simultaneous detection of DNA damage/repair by double-label technique Following the arrest of semiconservative D N A synthesis by S-phase cells, HL-60 cells were damaged by nitrogen-mustard (mechlorethamine) and incubated with [3H]Tdr subsequently to detect D N A repair by unscheduled DNA synthesis (UDS). Processing such samples by autoradiography revealed the presence of black grains in almost 90% of the cells. However, occasional cells were noted which were negative for grains, i.e. showed no repair. Whether such cells
A . RAZA et al.
12
TABLE 1. DETECTION OF SINGLE-STRANDED D N A DAMAGE IN H L - 6 0 CELLS USING MONOCLONAL ANTI-THYMIDINE ANTIBODY
Detection by MoAb20B7 (%)
No drug No drug + HCI DNR 5 Ixg/ml DNR 10 ~tg/ml
NEG
1+
2+
Karyorrhectic
Total+
100 0 26.5 16.5
0 0 38 18.5
0 100 33 43.5
0 0 2.5 21.5
0 100 73.5 83.5
DNR, daunomycin; MoAb20B7, monoclonal anti-thymidine antibody; HCI, hydrochloric acid.
showed no repair because they were not damaged by daunomycin or were so badly damaged that they could not undertake repair, are possibilities which can not be distinguished from each other by only using UDS. However, when MoAb20B7 and UDS were used simultaneously (Fig. 3), we found that most of the cells which showed no grains were strongly positive with MoAb20B7. Only an occasional cell with no grains demonstrated no reaction with MoAb20B7. Thus, by using the doublelabel technique, we were able to identify the reason why a cell showed no repair (no damage versus extensive damage). 4. In vivo detection of single-stranded DNA damage in patients receiving daunomycin-containing combination chemotherapy Figure 4 shows the blast cells processed by MoAb20B7 from PB of an AML patient 24 h after starting chemotherapy. Table 2 summarizes these data on the four AML patients being reported here. In every case, a marked increase in the percentage of damaged cells was noted at 24 h. This damage ranged from 1+ to karyorrhexis in individual cells.
DISCUSSION Treatment of cancer would substantially improve if we could avoid giving chemotherapy which has little or no effect on the tumor, but which frequently produces life-threatening side-effects. The target of most anti-cancer agents is DNA. Exposure of cancer cells to chemotherapeutic agents such as the anthracyclines, etoposide, cisplatin, and the alkylating agents produce single-strand breaks, double-strand breaks, and/or cross-linking of DNA [21,22]. The mechanism by which DNA damage is produced varies from agent to agent. Daunorubicin, cisplatin, and the alkylating agents directly interact with DNA [23] while etoposide and mAmsa produce DNA damage
indirectly by inhibiting the action of topoisomerase II [24]. In some cases, doxorubicin has been shown to cause damage by inducing the production of free radicals in the cell. A simple test, which could reliably estimate the presence or absence of DNA damage is sorely needed. Complex and highly sophisticated tests such as alkaline elution and equilibrium sedimentation are impractical since they cannot be easily applied to clinical situations. Several new approaches have recently been described in the literature. One of these recently described methods is from our laboratory and utilized a modified Southern blotting technique to detect double-stranded DNA damage [25]. To quantitate single-stranded DNA damage, two approaches have previously been described. The first is based upon production of antibodies to damaged DNA. Lucas in 1972 [10] and Frankfurt in 1987 [11] have been successful in using ultraviolet light (UV) damaged DNA (Lucas) and nitrogen-mustard damaged DNA (Frankfurt) to produce such antibodies. Lucas showed that the antigenic determinants in their experiments were thymine dimers. They produced antisera containing antibodies reactive with UV-irradiated DNA based on the assumption that during early repair, a segment of DNA which is approximately 200 nucleotides in length is excised, exposing pyrimidine dimers on the opposite strand. Their assumption was correct since they were able to achieve labeling of UV-induced DNA lesions using the antisera thus prepared, which was shown to contain antibodies to thymidine. However, their antibodies were polyclonal. Frankfurt [ 11] produced a monoclonal antibody to nitrogen-mustard damaged DNA and was able to use this antibody to detect single-stranded DNA damage from individual Hela cells exposed to nitrogen mustard. A different approach to detect single-stranded DNA damage was used by Beiser et al. who produced antibodies to guanosine with the concept that removal of damaged DNA strand would expose the nucleotides on the intact opposite strand which could
D N A damage from individual cells
13 n
m FIG. 2. (a) HL-60 cells which were neither treated by HC1 nor daunomycin, but processed by the monoclonal antithymidine antibody 20B7 show no positive labeling. (b) HL-60 cells whose D N A was denatured by HC1 prior to treatment with MoAb20B7 show that every cell is brightly reactive with the antibody. (c) HL-60 cells incubated with 5 ~tg/ml of daunomycin for 24 h and then treated by MoAb20B7 show varying degrees of reactivity with the antibody.
14
A. RAZAet
al.
FIG. 3. "Double-labeled" HL-60 cells which combine the anti-thymidine antibody treatment and unscheduled D N A synthesis. These cells were damaged by incubations with nitrogen-mustard. The two very brightly positive cells (dark brown-red) do not show any grains over their nuclei, indicating excessive damage as the reason for absence of repair. F16. 4. Freshly obtained human leukemic cells from a patient with AML undergoing remission induction chemotherapy. Once again, this peripheral blood sample obtained 24 h after the start of araC and daunomycin shows varying degrees of reactivity of the cells with anti-thymidine antibody.
15
D N A damage from individual cells T A B L E 2. IN VIVO DETECTION OF SINGLE-STRANDED DNA DAMAGE IN CHEMOTHERAPY
AML PATIENTS
RECEIVING
Detection by MoAb20B7 (%) Case
Diagnosis
Sample
NEG
1+
2+
Karyorrhectic
Total +
1
AML
Pre PB Post PB
90 19
8 78
0 0
2 3
10 81
2
AML
Pre PB Post PB
95 4
4 72
1 8
0 16
5 96
3
AML
Pre PB Post PB
94 29
3 65
0 2
3 4
6 71
4
AML
Pre PB Post PB
95 5
4 90
0 5
1 0
5 95
AML, acute myeloid leukemia; Pre PB, peripheral blood obtained prior to starting remission induction chemotherapy; Post PB, peripheral blood obtained 24 h after starting chemotherapy.
then be detected by the anti-nucleotide antibody [27]. However, since guanosine is not unique to the DNA, extensive treatment of all samples with RNAse had to be undertaken prior to using the anti-guanosine antibody. Furthermore, this was a polyclonal antibody and therefore, its specificity was not as good as that of a monoclonal antibody. We have developed the current method based on the same concept, but using thymidine as a target. Using the monoclonal anti-thymidine clone 20B7, we have been able to successfully develop a simple and reliable assay which detects single-stranded DNA damage with alacrity. Proof that our antibody is indeed detecting this phenomenon in cells must remain circumstantial. Several observations made on the experiments being reported here, however, indirectly support our assumption. Firstly, our carefully conducted positive and negative controls are consistently reproducible in that cells whose DNA is denatured by acid treatment are brightly positive, while samples where either no MoAb20B7 or some other unrelated antibody (anti-bromodeoxyuridine antibody) was used are universally negative. Secondly, daunomycin treated cells demonstrated varying degrees of reactivity with the monoclonal anti-thymidine antibody implying the binding of the antibody to exposed strands of DNA, which would be consistent with our hypothesis. Moreover, this reactivity showed a "dose response curve" in that labeling was noted to be much brighter with escalating doses of daunomycin, and the morphological appearance of cells at higher doses clearly indicated chemotherapy induced damage even to the point of karyorrhexis. Thirdly, the double-labeled slides which simultaneously measured damage caused by nitrogen-mustard and repair of the damage by
unscheduled DNA synthesis, showed that the cells undergoing repair (grains) were lightly labeled (less damage) compared to most of the cells showing no repair (no grains), which were brightly labeled (extensive damage). Finally, the viability experiments conducted using trypan-blue exclusion demonstrated that DNA damage as detected by the MoAb20B7 antibody is not synonymous with cell death. In fact, despite maintaining viability in the majority of cells, increasing degree of reactivity with the antibody was observed with escalating doses of daunomycin. One intriguing possibility which remains unanswered in the present study is whether our monoclonal 20B7 will be able to detect exposed thymidine bases present in open replication forks of cells undergoing DNA synthesis. Indeed, Beiser et al. were able to perform labeling index (LI) determinations from tissue specimens by using polyclonal anti-guanosine antibodies [26, 27]. Using standard methods described here, MoAb20B7 is unable to identify Sphase cells. The observation that our antibody does not react with the replicating forks in S-phase cells may be due to the fact that the length of actually exposed replicons in an S-phase cell is smaller [28, 29] than repairing DNA segments (50-200 nucleotides versus 200 nucleotides, respectively), or that the replicons are covered by proteins necessary for DNA synthesis to continue (DNA polymerase, histones etc.), thereby obscuring the bases while the bases in repairing segments are not covered by proteins. The latter problem may be overcome by employing mild detergents to remove proteins, which to some extent may have happened in the experiments conducted by Beiser et al. who used RNAse treatment prior to reacting tissue specimens with their anti-guanosine
16
A. RAZAet al.
antibodies and were able to detect S-phase cells [27]. It is worth noting here that Frankfurt [11] was also unable to detect S-phase cell using his monoclonal antibody to nitrogen-mustard d a m a g e d D N A . Finally, the most important aspect of the work being reported here is the application of this technique to clinical settings. As demonstrated in this paper, we were successful in detecting a difference in the percentage as well as degree of labeling observed in freshly obtained h u m a n leukemic cells prior to and following treatment with daunomycin containing regimens in A M L patients. If this observation is further substantiated in larger n u m b e r of patients, then one can conceive using this simple and elegant technique to m a k e therapeutic decisions in patients undergoing chemotherapy. For example, if minimal or no damage is observed 24 h after one dose of daunomycin, then alternate therapies must be considered so that potentially lethal side-effects of the drug can be avoided which would invariably occur even in the absence of benefit to the patient. Moreover, this assay can be used to determine the reversal of the multidrug resistance ( M D R ) gene in patients with overexpression of the P170 m e m b r a n e protein [30]. In fact, our early experiments indicate that incubating cells with daunorubicin and calcium channel blockers such as verapamil in vitro substantially increases the D N A damage observed in individual cells. These experiments will be reported elsewhere (manuscript in preparation). Suffice it to say that a point in time where almost no methods are available to provide information regarding D N A damage in individual cells in a p r o m p t enough fashion to allow for therapeutic intervention, the monoclonal thymidine antibody m a y b e c o m e an extremely useful tool for improving treatment in cancer patients.
Acknowledgements--The authors would like to thank
4. Raza A., Gezer S., Anderson J., Browman G., Goldberg J. et al. (1989) Recognition of a subgroup of patients with acute myeloid leukemia (AML) who will not respond to therapy with high dose cytosine arabinoside using bromodeoxyuridine and tritiated araC in vitro. Proc. Am. Soc. Clin. Oncol. Abst. 778. 5. Kohn K. W. (1981) Measurement of strand breaks and crosslinks by alkaline elution. In D N A Repair: A Lab Manual o f Research Procedures (Friedberg et al. , Eds), Vol. 1 (Part B), p. 379--402. 6. Aaij C. & Borst P. (1972) The gel electrophoresis of DNA. Biochem. Biophys. Acta 269, 192. 7. Hanawalt P. C. (1977) D N A repair process. A n overview in Cellular Senescence and Somatic Cell Genetics
8. 9. 10. 11. 12.
13.
14.
15.
16.
Ms Angela Cuppone for excellent secretarial assistance. This work was supported by National Cancer Institute Grants CA-28734 and CA-41285-03.
17.
REFERENCES
18.
1. Preisler H. D., Rustum Y., Henderson E. S. & Bjornsson S. et al. (1979) Treatment of acute nonlymphocytic leukemia: Use of anthracycline-cytosine arabinoside induction therapy and comparison of two maintenance regimens. Blood 53, 455. 2. Smets L. A. & Homan-Blok J. (1985) S-Phase cells of the leukemic cell cycle sensitive to 1-B-D-arabinofuranosylcytosine at a high dose level. Cancer Res. 45, 3113. 3. Spriggs D., Robbins G., Ohno Y. & Kufe D. (1987) Detection of 1-B-D-arabinofuranosylcytosine incorporation into DNA in vivo. Cancer Res. 47, 6532.
19. 20. 21.
22.
(Nichold W. & Murphy D. Eds), pp. 1-18. Pointer R. B. & Cleaver J. E. (1969) Repair replication, unscheduled DNA synthesis and repair of mammalian DNA. Rad. Res 37, 451. Speigler P. & Norman A. (1969) Kinetics of unscheduled DNA synthesis induced by ionizing radiation in human lymphocytes. Rad. Res. 39, 400. Lucas C. J. (1972) Immunological demonstration of the disappearance of pyrimidine dimers from nuclei of cultured human cells. Exp. Cell Res. 74, 480. Frankfurt O. S. (1987) Detection of DNA damage in individual cells by flow cytometric analysis using antiDNA monoclonal antibody. Expt. Cell Res. 170, 369. Beisker W. & Hittelman W. N. (1988). Measurement of the kinetics of DNA repair synthesis after UV irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry. Expt. Cell Res. 174, 156. Dalton W. D., Ahearn J. M. Jr, McCredie K., Freireich E. J., Stass S. A. & Trujillo J. M. (1988) HL-60 line was derived from a patient with FAB-M2 and not FAB M3. Blood 71, 242. Erlanger B. F. & Beiser S. M. (1971) Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc. Nat. Acad. Sci. U.S.A. 52, 68. Kearney J. F., Radbruch A., Liesegang B. & Rajewsky K. (1979) New mouse myeloma cell line that has lost immunoglobulin expression but permits construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1548. Kohler G. & Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Getter M., Margules H. & Scharff M. D. (1977) A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Gen. 3, 231. Claflin L. & Williams K. (1978) Mouse myeloma-spleen cell hybrids: Enhanced hybridization frequencies and rapid screening procedures. Current topics Microbiol. Immunol. 81, 107. Galfre G. & Milstein C. (1981) Preparation of monoclonal antibodies. Strategies and procedures. Meth. lmmunol. 73, 1. Sommer J. R. (1977) To cationize glass. J. Cell Biol. 75, 245. Ross W. E., Glaubiger D. & Kohn K. W. (1979) Qualitative and quantitative aspects of intercalatorinduced DNA strand breaks. Biochem. Biophys. Acta 562, 41. Long B. H., Musial S. T. & Brattain M. G. (1985)
DNA damage from individual cells
23. 24.
25. 26.
Single and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide. Cancer Res. 45, 3106. Potmesil M., Israel M. & Silber R. (1984) Two mechanisms of adriamycin-DNA interaction in L1210 cells. Biochem. Pharm. 33, 3137. Ross W. E., Rowe T., Glisson B., Yalowich J. & Lin L. (1984) Role of topoisomerase. II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Res. 44, 5857. Kawamura M. & Preisler H. D. (1989) A new approach to the detection of DNA damage. Leukemia Res. 13, 391. Erlanger B. F. (1972) Nucleic acid reactive antibodies
17
specific for nucleosides and nuclotides. Acta Endocrin. Suppl. 168, 206. 27. Beiser S. M. etal. (1964) Purine and pyrimidine specific antibodies: Precipitation with developed DNA. Nature 203, 1381. 28. Walker I. G. & Th'ng J. P. H. (1982) Excision-repair patch size in DNA from human KB cells treated with UV-light, or methyl methanesulfonate. Mutat. Res. 105, 277. 29. Cleaver J. E. (1974) Repair processes for photochemical damage in mammalian cells. Adv. Radiat. Biol. 4, 1.
30. Moscow J. A. & Cowan K. H. (1988) Multidrug resistance. J. Nat. Cancer Inst. 80, 14.
0145-2126/91 $3.00 + .00 Pergamon Press plc
D E T E C T I O N OF SINGLE-STRANDED DNA D A M A G E USING M O N O C L O N A L ANTI-THYMIDINE ANTIBODY AZRA RAZA,* IQBAL MEHDI,* WEN JEN Guo,* NAVEED YOUSUF,* MARGARET MASTERSON,~" SALVADOREMIRTO,:~ LAWRENCEE. MOTYKA§ and GEORGE L. MAYERS§ *Barrett Cancer Center, University of Cincinnati and tChildren's Hospital Medical Center of Cincinnati, 231 Bethesda Avenue, ML 562, Rm 6367, Cincinnati, OH 45208, U.S.A., ~:Ospedale "V. Cervello", Divisione Di Ematoiogia, Palermo, Italy, §Roswell Park Memorial Institute, Department of Molecular Immunology, 666 Elm Street, Buffalo, NY 14263, U.S.A. (Received 27 October 1989. Revision accepted 6 June 1990) Abstract--A method to detect single-stranded DNA damage from individual cells has been developed
using a monoclonal anti-thymidine antibody (MoAb20B7). Initially, HL-60 cells were incubated with daunomycin at different concentrations, and processed by MoAb20B7. While 73.5% of the cells incubated with 5 ~tg/ml of daunomycin for 24 h reacted positively with MoAb20B7, 83.5% cells at 10 ~tg/ml daunomycin dose were positive. Next, this method was combined with unscheduled DNA synthesis to simultaneously measure repair and damage from individual cells. Finally, patients with acute myeloid leukemias were studied before and 24 h after therapy with a daunomycin containing regimen. In vivo damage could be determined in a prompt fashion. Key words: DNA damage, DNA repair, monoclonal anti-thymidine antibody, AML.
INTRODUCTION THE MAINSTAYof therapy in acute myeloid leukemia (AML) is a combination of cytosine arabinoside (araC) and an anthracycline such as daunomycin [1]. An accurate prediction of response to this regimen in individual cases would substantially improve treatment results since the delivery of noxious agents with little or no benefit could be avoided. A number of in vitro assays have been shown to be useful for the S-phase specific agent araC because of its ability to become incorporated in the newly synthesized strand of DNA and exert its potentially lethal effect on the cell by inhibiting further DNA synthesis [2-4]. DNA damaging agents such as the anthracyclines present a more complicated problem, since these agents are intercalators of DNA rather than becoming incorporated into DNA. Thus, their effects on the cell can only be measured indirectly. While a number of highly Abbreviations: MoAb2OB7, monoclonal anti-thymidine antibody; AML, acute myeloid leukemia; araC, cytosine arabinoside; UDS, unscheduled DNA synthesis; FCS, fetal calf serum; PBS, phosphate buffered saline; PB, peripheral blood; BM, bone marrow aspirate; HCI, hydrochloric acid; LI, labeling index; UV, ultraviolet light; MDR, multidrug resistance. Correspondence to: Dr Azra Raza, Director, Hematologic Oncology, Barrett Cancer Center, University of Cincinnati, 231 Bethesda Avenue, Room 330, ML 508, Cincinnati, OH 45267, U.S.A.
sophisticated methods to measure DNA damage have been developed in the past, most have remained confined to a few laboratories by virtue of their complexity. In addition, these methods only provided information on the population of cells as a whole rather than the individual cell [5-9]. More recently, assays have been developed to detect DNA damage from individual cells. These assays are based on antibodies produced against damaged DNA segments [10-121 . We would like to describe the development of a relatively similar method to detect single-stranded DNA damage which by virtue of its simplicity, may become an important assay for prediction of response to anthracyclines in AML. A monoclonal antibody to thymidine (MoAb20B7) was develope d based upon the hypothesis that during the repair of singlestranded damaged DNA, thymidine bases on the opposite, non-damaged strand would be available for reaction with the antibody. Thus, while in a nondamaged cell, the thymidine antibody would only react with the incorporated bases following denaturation and unwinding of the helix, single-stranded damaged DNA would be reactive with the antibody without necessitating denaturation. Moreover, this method was combined with conventional autoradiography to detect unscheduled DNA synthesis (UDS), thereby allowing us to measure DNA damage (antithymidine antibody) and repair (UDS) simultaneously from individual cells.
A. RAZAet al.
10 MATERIALS
AND METHODS
Initial work on the detection of single-stranded D N A damage and repair was conducted on the HL-60 tissue culture cell line. The characteristics of this cell line have been described at length elsewhere [13]. Briefly, it was originally derived from a patient with acute myeloid leukemia and now grows in RPMI 1640 cell culture medium supplemented with 10% fetal calf serum (FCS) and penicillin and streptomycin. In order to simplify this section, methods will be described under the following sub-headings.
1. Preparation of monoclonal anti-thymidine antibody 20B7 Hybridomas were prepared by the fusion of spleen cells from B A L B / c mice immunized with a 5-methyluridinebovine gammaglobulin conjugate [14] and P3X63-Ag8.653 myeloma cells [15] using standard procedures [16-19]. Hybridoma 20B7 was propagated in pristane treated BALB/c mice and the monoclonal anti-thymidine antibodies were isolated from the ascitic fluid. All antibodies used in these studies were affinity purified on an AHsepharose 4B-5-methyluridine affinity column. Monoclonal anti-thymidine antibody 20B7 was characterized in radioimmunoassay and shown to be highly specific for thymidine and to have no specificity for guanosine, adenosine, cytidine and to have only minimal reactivity with uridine ( 1000fold less than thymidine).
2. Detection of single-stranded MoAb2OB7
DNA
damage using
HL-60 cells in the concentration of 1 x 106/ml were incubated with daunomycin at doses of 5 ~tg and 10 ~tg/ml for 24 h. A control group of cells with no drug was also used. At the end of 24 h of incubation, ceils were washed thrice with phosphate buffered saline (PBS) at 1000 g for 5 min each and then placed on alcian-blue coated coverslips. Alcian-blue was used to cationize glass, which allows the cells to be evenly distributed throughout the coverslip and cell loss during processing is minimized due to the firm adherence produced by the electrical charge [20]. Cells were fixed in 70% ethanol for 10 min and processed by the indirect immunoperoxidase method using MoAb20B7 as described below. After fixation, the coverslips were washed in Tris buffered saline (TBS) and placed in a jar containing 100 ml of absolute methanol and 25 ml of 3% HzO2 for 20 min at room temperature (RT). This step would remove any endogenous peroxidase from cells, which was particularly important when samples of A M L patients were being studied. The coverslips were washed again in TBS and then three coverslips per group were processed by the MoAb20B7. In the first "positive control" coverslip of each group, D N A was denatured by a 20 min treatment with 4 N HC1 at RT, while no HC1 treatment was used for the actual experimental group. Coverslips from both the control and the experimental groups were then exposed to Tris buffer containing 0.01% bovine serum albumin (TrisBSA) for l h at 37°C in 5% CO2. MoAb20B7 diluted 1 : 2000 with PBS and 0.05% nonidet 40 (NP-40) was pipetted onto each coverslip for one hour at RT. Plain PBS and NP-40 without MoAb20B7 was pipetted onto the second "negative controls", i.e. cells which were treated with
daunomycin, and processed by the whole immunoperoxidase method save for the use of the primary antibody MoAb20B7. Coverslips were rinsed in PBS/Triton 100 (0.1%) for 1 h and exposed to peroxidase conjugated rabbit anti-mouse antibody (P161 from D A K O ) diluted 1:100 with TBS/4% human serum for 30 min at RT. The coverslips were rinsed in TBS followed by a 20 min exposure to freshly prepared and filtered ethylamine carbazine (AEC) at 37°C in 5% CO2. A final rinse in cold tap water was carried out and the coverslips were mounted onto glass slides with immunomount and examined under a light microscope. Any cell in which the nucleus stained anywhere from light pink to dark brown (or red) was counted as positive. At least 500-2000 cells were examined from randomly chosen fields from each coverslip and the results were presented as the percentage of positive cells.
3. Simultaneous detection of DNA damage and repair by the "double-label" technique combining anti-thymidine antibody with unscheduled DNA synthesis HL-60 cells at a concentration of 1 x 106/ml were incubated with 10-1 M hydroxyurea (HU) (Sigma Chemicals) for 2 h at 37°C in order to arrest semi-conservative D N A synthesis by S-phase cells [8]. In the last hour of the incubation, 100 ~tg of mechlorethamine/ml was added to the suspension in order to produce D N A damage. The cells were then washed thrice with RPMI 1640 and resuspended in RPMI 1640 cell culture medium at a concentration of 5 x 106 cells/ml. Freshly prepared HU at a dose of 10- l M was added to the cells along with 20 ~tl/ml of [3H]Tdr (specific activity 60-90 Ci/mmole, ICN Radiochemicals). After 2h, the cells were washed with nonradiolabeled thymidine thrice and once with PBS and placed on alcian blue coated coverslips. The coverslips were processed by the MoAb20B7 as described in Section 2 and then coated with NTB2 nuclear track emulsion (Eastman Kodak) at 42°C. The coverslips were allowed to dry completely and stored in the dark at - 6 0 to -80°C for 10 days. Kodak D-19 developer was used to develop the coverslips which were then fixed and mounted on glass slides with immunomount. Any cell with at least five silver grains was considered as positively labeled by [3H]Tdr, and ceils which stained anywhere from light pink to dark brown or red were considered as positive for MoAb20B7.
4. Detection of in vivo DNA damage in A M L patients undergoing chemotherapy with daunomycin as part of the regimen Four patients with A M L were also studied for the application of this technique to detect D N A damage induced in vivo by daunomycin. Peripheral blood (PB) and/or bone marrow (BM) aspirate samples were drawn into glass syringes containing 2% sodium citrate and light density cells were recovered following Ficoll-Hypaque separation. Cells were obtained from individual patients prior to starting remission induction chemotherapy (time 0) and serially therafter (4, 24, 48 h) following start of daunomycin containing chemotherapy combination. Following separation on the density-gradient, the mononuclear cells were placed on alcian-blue coated coverslips and processed by the single-label methodology described in Section 2. Please note that for every sample processed, two "controls" were prepared along with the "experimental" group. The first was a "positive" control in which D N A of every cell was denatured by using hydrochloric acid (HC1) so that when
DNA damage from individualcells I00
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FIo. 1. Fine specificity of monoclonal anti-thymidine antibody 20B7. This antibody shows the highest specificity for thymidine with some reactivity with the thymidine analogues 5-bromouridine and 5-iodouridine. This antibody does not react with the natural nucleosides guanosine, adenosine and cytidine and only weakly with uridine (1000fold less than thymidine).
MoAb20B7 was used following this treatment, every cell reacted positively with the antibody (since every cell contains thymidine). A "negative" control was accomplished in two parts. Firstly, control cells were processed exactly as the experimental group except that the MoAb20B7 was left out of the procedure. Secondly, an unrelated monoclonal antibody such as anti-bromodeoxyuridine which should not react with the cells being used was added in place of MoAb20B7. RESULTS In order to maintain clarity, this section will also be subdivided into separate sections.
1. Specificity of the thymidine antibody Monoclonal antibody 20B7 is one of large number of anti-thymidine antibodies whose preparation has been described in detail elsewhere (L,E.M. Ph.D. Thesis, manuscript in preparation). The fine specificity of MoAb20BT, which was isolated by affinity chromatography, has been evaluated in an R I A using thymidine, guanosine, adenosine, cytidine, uridine, 5-bromouridine, 5-iodouridine, and 5-fluorouridine as inhibitors. The results are shown in Fig. 1 which shows that this antibody does not have any specificity for guanosine, adenosine, or cytidine, and only weakly binds uridine (1000-fold weaker than thymidine). The antibody does show some weak affinity for 5-bromouridine and 5-iodouridine, the two thymidine analogues that can be incorporated into DNA in place of thymidine. In addition, in the immunocytochemical assays the binding of 20B7 can be completely inhibited by the addition of 100 ~tl of 1 x 10 - 4 M thymidine, but not by lO0~tl of 1 x 10 -v M thymidine.
11
2. Detection of single-stranded DNA damage produced by daunomycin HL-60 cells which were incubated with daunomycin at concentrations varying from 5 to 10 ~g/ml for 24 h and then processed by MoAb20B7 are shown in Figs 2 (a-c). Figure 2a represents the "negative" control where HL-60 cells were processed by the entire indirect immunoperoxidase method, except for the deletion of MoAb20B7, so that every cell is "white" or non-reactive. Similar results were obtained from the other two "negative" control groups where no HCI and no daunomycin were used, but the entire immunoperoxidase method using MoAb20B7 was performed or where anti-bromodeoxyuridine was used instead of MoAb20B7. Figure 2b is the "positive" control in which D N A was denatured by treatment with 4 N HC1 so that every cell is strongly reactive with MoAb20B7, demonstrating deep-red nuclear staining with peroxidase. Figure lc is the actual "experimental" group in which cells incubated with 5 ~tg/ml of daunomycin for 24 h were processed by MoAb20B7 and varying degrees of reactivity was noted in individual cells. Table 1 summarizes the single label data and is an average of two separate experiments. Column 1 describes the negative and positive controls first, which were noted to be universally negative and positive, for each experimental group, respectively. It can be seen that in the actual experimental group, there was an increasing percentage of cells positively reactive with MoAb20B7 in both the concentrations of daunomycin used. In the 5 ~tg/ml group, 26.5% cells were negative, 38% were 1+ reactive while 33% cells were brightly positive or 2+ reactive. In addition, 2.5% cells in this group were clearly karyorrhectic and their nuclei were noted to be undergoing fragmentation. Thus, in this group, a total of 73.5% cells were found to be reactive with the MoAb20B7. In the 10 ~tg/ml group, 16.5% cells were completely negative, 18.5% were 1 + positive, 43.5% were 2+ positive and 21.5% were karyorrhectic, giving a total of 83.5% cells reactive with the monoclonal antibody.
3. Simultaneous detection of DNA damage/repair by double-label technique Following the arrest of semiconservative D N A synthesis by S-phase cells, HL-60 cells were damaged by nitrogen-mustard (mechlorethamine) and incubated with [3H]Tdr subsequently to detect D N A repair by unscheduled DNA synthesis (UDS). Processing such samples by autoradiography revealed the presence of black grains in almost 90% of the cells. However, occasional cells were noted which were negative for grains, i.e. showed no repair. Whether such cells
A . RAZA et al.
12
TABLE 1. DETECTION OF SINGLE-STRANDED D N A DAMAGE IN H L - 6 0 CELLS USING MONOCLONAL ANTI-THYMIDINE ANTIBODY
Detection by MoAb20B7 (%)
No drug No drug + HCI DNR 5 Ixg/ml DNR 10 ~tg/ml
NEG
1+
2+
Karyorrhectic
Total+
100 0 26.5 16.5
0 0 38 18.5
0 100 33 43.5
0 0 2.5 21.5
0 100 73.5 83.5
DNR, daunomycin; MoAb20B7, monoclonal anti-thymidine antibody; HCI, hydrochloric acid.
showed no repair because they were not damaged by daunomycin or were so badly damaged that they could not undertake repair, are possibilities which can not be distinguished from each other by only using UDS. However, when MoAb20B7 and UDS were used simultaneously (Fig. 3), we found that most of the cells which showed no grains were strongly positive with MoAb20B7. Only an occasional cell with no grains demonstrated no reaction with MoAb20B7. Thus, by using the doublelabel technique, we were able to identify the reason why a cell showed no repair (no damage versus extensive damage). 4. In vivo detection of single-stranded DNA damage in patients receiving daunomycin-containing combination chemotherapy Figure 4 shows the blast cells processed by MoAb20B7 from PB of an AML patient 24 h after starting chemotherapy. Table 2 summarizes these data on the four AML patients being reported here. In every case, a marked increase in the percentage of damaged cells was noted at 24 h. This damage ranged from 1+ to karyorrhexis in individual cells.
DISCUSSION Treatment of cancer would substantially improve if we could avoid giving chemotherapy which has little or no effect on the tumor, but which frequently produces life-threatening side-effects. The target of most anti-cancer agents is DNA. Exposure of cancer cells to chemotherapeutic agents such as the anthracyclines, etoposide, cisplatin, and the alkylating agents produce single-strand breaks, double-strand breaks, and/or cross-linking of DNA [21,22]. The mechanism by which DNA damage is produced varies from agent to agent. Daunorubicin, cisplatin, and the alkylating agents directly interact with DNA [23] while etoposide and mAmsa produce DNA damage
indirectly by inhibiting the action of topoisomerase II [24]. In some cases, doxorubicin has been shown to cause damage by inducing the production of free radicals in the cell. A simple test, which could reliably estimate the presence or absence of DNA damage is sorely needed. Complex and highly sophisticated tests such as alkaline elution and equilibrium sedimentation are impractical since they cannot be easily applied to clinical situations. Several new approaches have recently been described in the literature. One of these recently described methods is from our laboratory and utilized a modified Southern blotting technique to detect double-stranded DNA damage [25]. To quantitate single-stranded DNA damage, two approaches have previously been described. The first is based upon production of antibodies to damaged DNA. Lucas in 1972 [10] and Frankfurt in 1987 [11] have been successful in using ultraviolet light (UV) damaged DNA (Lucas) and nitrogen-mustard damaged DNA (Frankfurt) to produce such antibodies. Lucas showed that the antigenic determinants in their experiments were thymine dimers. They produced antisera containing antibodies reactive with UV-irradiated DNA based on the assumption that during early repair, a segment of DNA which is approximately 200 nucleotides in length is excised, exposing pyrimidine dimers on the opposite strand. Their assumption was correct since they were able to achieve labeling of UV-induced DNA lesions using the antisera thus prepared, which was shown to contain antibodies to thymidine. However, their antibodies were polyclonal. Frankfurt [ 11] produced a monoclonal antibody to nitrogen-mustard damaged DNA and was able to use this antibody to detect single-stranded DNA damage from individual Hela cells exposed to nitrogen mustard. A different approach to detect single-stranded DNA damage was used by Beiser et al. who produced antibodies to guanosine with the concept that removal of damaged DNA strand would expose the nucleotides on the intact opposite strand which could
D N A damage from individual cells
13 n
m FIG. 2. (a) HL-60 cells which were neither treated by HC1 nor daunomycin, but processed by the monoclonal antithymidine antibody 20B7 show no positive labeling. (b) HL-60 cells whose D N A was denatured by HC1 prior to treatment with MoAb20B7 show that every cell is brightly reactive with the antibody. (c) HL-60 cells incubated with 5 ~tg/ml of daunomycin for 24 h and then treated by MoAb20B7 show varying degrees of reactivity with the antibody.
14
A. RAZAet
al.
FIG. 3. "Double-labeled" HL-60 cells which combine the anti-thymidine antibody treatment and unscheduled D N A synthesis. These cells were damaged by incubations with nitrogen-mustard. The two very brightly positive cells (dark brown-red) do not show any grains over their nuclei, indicating excessive damage as the reason for absence of repair. F16. 4. Freshly obtained human leukemic cells from a patient with AML undergoing remission induction chemotherapy. Once again, this peripheral blood sample obtained 24 h after the start of araC and daunomycin shows varying degrees of reactivity of the cells with anti-thymidine antibody.
15
D N A damage from individual cells T A B L E 2. IN VIVO DETECTION OF SINGLE-STRANDED DNA DAMAGE IN CHEMOTHERAPY
AML PATIENTS
RECEIVING
Detection by MoAb20B7 (%) Case
Diagnosis
Sample
NEG
1+
2+
Karyorrhectic
Total +
1
AML
Pre PB Post PB
90 19
8 78
0 0
2 3
10 81
2
AML
Pre PB Post PB
95 4
4 72
1 8
0 16
5 96
3
AML
Pre PB Post PB
94 29
3 65
0 2
3 4
6 71
4
AML
Pre PB Post PB
95 5
4 90
0 5
1 0
5 95
AML, acute myeloid leukemia; Pre PB, peripheral blood obtained prior to starting remission induction chemotherapy; Post PB, peripheral blood obtained 24 h after starting chemotherapy.
then be detected by the anti-nucleotide antibody [27]. However, since guanosine is not unique to the DNA, extensive treatment of all samples with RNAse had to be undertaken prior to using the anti-guanosine antibody. Furthermore, this was a polyclonal antibody and therefore, its specificity was not as good as that of a monoclonal antibody. We have developed the current method based on the same concept, but using thymidine as a target. Using the monoclonal anti-thymidine clone 20B7, we have been able to successfully develop a simple and reliable assay which detects single-stranded DNA damage with alacrity. Proof that our antibody is indeed detecting this phenomenon in cells must remain circumstantial. Several observations made on the experiments being reported here, however, indirectly support our assumption. Firstly, our carefully conducted positive and negative controls are consistently reproducible in that cells whose DNA is denatured by acid treatment are brightly positive, while samples where either no MoAb20B7 or some other unrelated antibody (anti-bromodeoxyuridine antibody) was used are universally negative. Secondly, daunomycin treated cells demonstrated varying degrees of reactivity with the monoclonal anti-thymidine antibody implying the binding of the antibody to exposed strands of DNA, which would be consistent with our hypothesis. Moreover, this reactivity showed a "dose response curve" in that labeling was noted to be much brighter with escalating doses of daunomycin, and the morphological appearance of cells at higher doses clearly indicated chemotherapy induced damage even to the point of karyorrhexis. Thirdly, the double-labeled slides which simultaneously measured damage caused by nitrogen-mustard and repair of the damage by
unscheduled DNA synthesis, showed that the cells undergoing repair (grains) were lightly labeled (less damage) compared to most of the cells showing no repair (no grains), which were brightly labeled (extensive damage). Finally, the viability experiments conducted using trypan-blue exclusion demonstrated that DNA damage as detected by the MoAb20B7 antibody is not synonymous with cell death. In fact, despite maintaining viability in the majority of cells, increasing degree of reactivity with the antibody was observed with escalating doses of daunomycin. One intriguing possibility which remains unanswered in the present study is whether our monoclonal 20B7 will be able to detect exposed thymidine bases present in open replication forks of cells undergoing DNA synthesis. Indeed, Beiser et al. were able to perform labeling index (LI) determinations from tissue specimens by using polyclonal anti-guanosine antibodies [26, 27]. Using standard methods described here, MoAb20B7 is unable to identify Sphase cells. The observation that our antibody does not react with the replicating forks in S-phase cells may be due to the fact that the length of actually exposed replicons in an S-phase cell is smaller [28, 29] than repairing DNA segments (50-200 nucleotides versus 200 nucleotides, respectively), or that the replicons are covered by proteins necessary for DNA synthesis to continue (DNA polymerase, histones etc.), thereby obscuring the bases while the bases in repairing segments are not covered by proteins. The latter problem may be overcome by employing mild detergents to remove proteins, which to some extent may have happened in the experiments conducted by Beiser et al. who used RNAse treatment prior to reacting tissue specimens with their anti-guanosine
16
A. RAZAet al.
antibodies and were able to detect S-phase cells [27]. It is worth noting here that Frankfurt [11] was also unable to detect S-phase cell using his monoclonal antibody to nitrogen-mustard d a m a g e d D N A . Finally, the most important aspect of the work being reported here is the application of this technique to clinical settings. As demonstrated in this paper, we were successful in detecting a difference in the percentage as well as degree of labeling observed in freshly obtained h u m a n leukemic cells prior to and following treatment with daunomycin containing regimens in A M L patients. If this observation is further substantiated in larger n u m b e r of patients, then one can conceive using this simple and elegant technique to m a k e therapeutic decisions in patients undergoing chemotherapy. For example, if minimal or no damage is observed 24 h after one dose of daunomycin, then alternate therapies must be considered so that potentially lethal side-effects of the drug can be avoided which would invariably occur even in the absence of benefit to the patient. Moreover, this assay can be used to determine the reversal of the multidrug resistance ( M D R ) gene in patients with overexpression of the P170 m e m b r a n e protein [30]. In fact, our early experiments indicate that incubating cells with daunorubicin and calcium channel blockers such as verapamil in vitro substantially increases the D N A damage observed in individual cells. These experiments will be reported elsewhere (manuscript in preparation). Suffice it to say that a point in time where almost no methods are available to provide information regarding D N A damage in individual cells in a p r o m p t enough fashion to allow for therapeutic intervention, the monoclonal thymidine antibody m a y b e c o m e an extremely useful tool for improving treatment in cancer patients.
Acknowledgements--The authors would like to thank
4. Raza A., Gezer S., Anderson J., Browman G., Goldberg J. et al. (1989) Recognition of a subgroup of patients with acute myeloid leukemia (AML) who will not respond to therapy with high dose cytosine arabinoside using bromodeoxyuridine and tritiated araC in vitro. Proc. Am. Soc. Clin. Oncol. Abst. 778. 5. Kohn K. W. (1981) Measurement of strand breaks and crosslinks by alkaline elution. In D N A Repair: A Lab Manual o f Research Procedures (Friedberg et al. , Eds), Vol. 1 (Part B), p. 379--402. 6. Aaij C. & Borst P. (1972) The gel electrophoresis of DNA. Biochem. Biophys. Acta 269, 192. 7. Hanawalt P. C. (1977) D N A repair process. A n overview in Cellular Senescence and Somatic Cell Genetics
8. 9. 10. 11. 12.
13.
14.
15.
16.
Ms Angela Cuppone for excellent secretarial assistance. This work was supported by National Cancer Institute Grants CA-28734 and CA-41285-03.
17.
REFERENCES
18.
1. Preisler H. D., Rustum Y., Henderson E. S. & Bjornsson S. et al. (1979) Treatment of acute nonlymphocytic leukemia: Use of anthracycline-cytosine arabinoside induction therapy and comparison of two maintenance regimens. Blood 53, 455. 2. Smets L. A. & Homan-Blok J. (1985) S-Phase cells of the leukemic cell cycle sensitive to 1-B-D-arabinofuranosylcytosine at a high dose level. Cancer Res. 45, 3113. 3. Spriggs D., Robbins G., Ohno Y. & Kufe D. (1987) Detection of 1-B-D-arabinofuranosylcytosine incorporation into DNA in vivo. Cancer Res. 47, 6532.
19. 20. 21.
22.
(Nichold W. & Murphy D. Eds), pp. 1-18. Pointer R. B. & Cleaver J. E. (1969) Repair replication, unscheduled DNA synthesis and repair of mammalian DNA. Rad. Res 37, 451. Speigler P. & Norman A. (1969) Kinetics of unscheduled DNA synthesis induced by ionizing radiation in human lymphocytes. Rad. Res. 39, 400. Lucas C. J. (1972) Immunological demonstration of the disappearance of pyrimidine dimers from nuclei of cultured human cells. Exp. Cell Res. 74, 480. Frankfurt O. S. (1987) Detection of DNA damage in individual cells by flow cytometric analysis using antiDNA monoclonal antibody. Expt. Cell Res. 170, 369. Beisker W. & Hittelman W. N. (1988). Measurement of the kinetics of DNA repair synthesis after UV irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry. Expt. Cell Res. 174, 156. Dalton W. D., Ahearn J. M. Jr, McCredie K., Freireich E. J., Stass S. A. & Trujillo J. M. (1988) HL-60 line was derived from a patient with FAB-M2 and not FAB M3. Blood 71, 242. Erlanger B. F. & Beiser S. M. (1971) Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc. Nat. Acad. Sci. U.S.A. 52, 68. Kearney J. F., Radbruch A., Liesegang B. & Rajewsky K. (1979) New mouse myeloma cell line that has lost immunoglobulin expression but permits construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1548. Kohler G. & Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Getter M., Margules H. & Scharff M. D. (1977) A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Gen. 3, 231. Claflin L. & Williams K. (1978) Mouse myeloma-spleen cell hybrids: Enhanced hybridization frequencies and rapid screening procedures. Current topics Microbiol. Immunol. 81, 107. Galfre G. & Milstein C. (1981) Preparation of monoclonal antibodies. Strategies and procedures. Meth. lmmunol. 73, 1. Sommer J. R. (1977) To cationize glass. J. Cell Biol. 75, 245. Ross W. E., Glaubiger D. & Kohn K. W. (1979) Qualitative and quantitative aspects of intercalatorinduced DNA strand breaks. Biochem. Biophys. Acta 562, 41. Long B. H., Musial S. T. & Brattain M. G. (1985)
DNA damage from individual cells
23. 24.
25. 26.
Single and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide. Cancer Res. 45, 3106. Potmesil M., Israel M. & Silber R. (1984) Two mechanisms of adriamycin-DNA interaction in L1210 cells. Biochem. Pharm. 33, 3137. Ross W. E., Rowe T., Glisson B., Yalowich J. & Lin L. (1984) Role of topoisomerase. II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Res. 44, 5857. Kawamura M. & Preisler H. D. (1989) A new approach to the detection of DNA damage. Leukemia Res. 13, 391. Erlanger B. F. (1972) Nucleic acid reactive antibodies
17
specific for nucleosides and nuclotides. Acta Endocrin. Suppl. 168, 206. 27. Beiser S. M. etal. (1964) Purine and pyrimidine specific antibodies: Precipitation with developed DNA. Nature 203, 1381. 28. Walker I. G. & Th'ng J. P. H. (1982) Excision-repair patch size in DNA from human KB cells treated with UV-light, or methyl methanesulfonate. Mutat. Res. 105, 277. 29. Cleaver J. E. (1974) Repair processes for photochemical damage in mammalian cells. Adv. Radiat. Biol. 4, 1.
30. Moscow J. A. & Cowan K. H. (1988) Multidrug resistance. J. Nat. Cancer Inst. 80, 14.
