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[CANCER RESEARCH 46, 2324-2329, May 1986] Cell Cycle-dependent Cytotoxicity of Alkylating Agents: Determination of Nitrogen Mustard-induced DNA Cross-Links and Their Repair in Chinese Hamster Ovary Cells Synchronized by Centrifugal Elutriation1 David Murray2 and Raymond E. Meyn Department of Experimental Radiotherapy, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030 ABSTRACT Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal vlut ria t¡onand treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antituntor agent. In agreement with previous studies, cell populations enriched in <;, were the most sensitive to HN2, and those enriched in late S phase-C-2 were more resistant, as determined by clonogenic assay. Although the variation in surviving fraction through the cell cycle in response to a single dose (3 UK/HII; 1.0 h) of HN2 was as great as a factor of 10, complete dose-response curves generated for the most sensitive and most resistant elutriator fractions indicated that such changes could be accounted for by a ratio of D»values of only 1.4. Cells synchronized by this same method were also analyzed for their relative levels of 11N 2induced DNA cross-linking using the sensitive technique of alkaline elution. There was no significant difference in the levels of either DNA interstrand or DNA-protein cross-links induced in the two elutriator fractions described above immediately after the HN2 treatment. When the amount of DNA cross-linking in the two fractions was measured 6 h after treatment, considerable repair had occurred; however, there was no measurable difference in the rate of repair of either type of cross-link (i.e., DNA interstrand and DNA-protein) in the different phases. Differ ences in DNA damage and repair processes could not therefore be resolved within the confidence limits of available assays and probably cannot account for the differential cytotoxicity of HN2 towards cells in the different cell cycle phases. INTRODUCTION Since the initial observation that the cytotoxicity of nitrogen and sulfur mustards depended on the position of the cell in the cell cycle at the time of treatment (1), the phase specificities of many bifunctional alkylating antitumor agents have been char acterized using a variety of cell lines and synchronization tech niques (for review, see Ref. 2). Despite this diversity of experi mental approaches, a consistent pattern has emerged: cells in M or early d are usually the most sensitive to such drugs, followed by those at the d-S-phase transition, whereas cells in mid to late S phase are the most resistant. The ultimate poten tial for clinically exploiting such characteristics depends on a more complete understanding of the relationships between these variations in cytotoxicity and biochemical effects of the drug in the different phases of the cell cycle. DNA is generally thought to be the principal target for these drugs, based pri marily on the results of studies with mutant bacterial cells that show extreme sensitivity to alkylating agents and an inability to remove alkylated bases from their genome (3, 4). Such observations have been extended to mammalian systems using genetically defined DNA repair-deficient mutants of CHO3 cells isolated by Thompson et al. (S) on the basis of their sensitivity to UV. These mutants are cross-sensitive to the antitumor drugs c/s-platinum and mitomycin C (6) and to the bifunctional alkylating agents melphalan and HN2 (2, 7), and they show an impaired ability to remove DNA cross-links produced by these drugs. The objective of the present study was to investigate the possible involvement of DNA damage and repair processes and in particular the formation and removal of DNA cross-links in the cell cycle-dependent response to a particular alkylating agent, HN2; specifically, the following questions have been addressed: (a) does the amount of HN2-induced DNA damage, or the capacity of cells to repair this damage, vary in different phases of the cell cycle? (b) Do such changes, if they occur, correlate with the measured cell cycle phase-dependency of the cytotoxicity of HN2? In order to address these questions, CHO cells were synchronized by centrifugal elutriation, a method that permits the rapid physical synchronization of cells in large enough numbers to allow the quantitation of DNA damage. DNA damage was measured using the sensitive alkaline elution technique developed by Kohn (8) which has the advantage that it allowed these investigations to be performed using sufficiently low concentrations of HN2 such that the molecular measure ments could be compared directly to the cytotoxicity. The results suggest that there is no significant difference in initial DNA damage induction in the different cell cycle phases and furthermore that any possible alterations in the repair of DNA damage in different phases of the cell cycle are too subtle to be resolved with statistical significance and are therefore unlikely to be a major determinant of the observed age-response pattern for HN2 cytotoxicity. MATERIALS AND METHODS Cell Culture Methods. CHO cells were maintained in exponential monolayer culture at 37*C in a humidified 5% Å’>2-95%air atmosphere in McCoy's 5A medium (Hsu's modification, Formula No. 78-5192; GIBCO, Grand Island, NY) supplemented with 15% FBS (Irvine Sci entific, Santa Ana, CA). Under these conditions, the cells had a 12- to 14 h doubling time and an 11.5-h generation time; the approximate durations of the individual phases were 2.5 (d), 6.75 (S), 1.75 (G2), and 0.5 h (M). Prior to elutriation, cells were harvested at 37°Cusing 0.025% trypsin containing DNase, 20 Mfi/nil. Trypsinization was quenched after 7 min by adding an equal volume of complete medium, and the cells were vigorously pipetted and syringed through a 22-gauge needle to minimize clumping. Light microscopy was used to inspect the quality of the single-cell suspensions. Centrifugal Elutriation. Cells were synchronized by the centrifugal Received 4/15/85; revised 1/15/86; accepted 2/3/86. elutriation method (9, 10), which exploits the fact that cells continu 1This investigation was supported by USPHS grant CA 23270 awarded by the ously increase in size as they progress through the cell cycle. This National Cancer Institute. 2 To whom requests for reprints should be addressed, at Department of method has several advantages (9) including the fact that it yields Experimental Radiotherapy, Box 66, M. D. Anderson Hospital and Tumor adequate synchrony for this application when compared with other Institute, 6723 Bertner Avenue, Houston TX 77030. techniques (10, 11). One disadvantage is that it does not allow charac 3The abbreviations used are: CHO, Chinese hamster ovary; HN2, nitrogen terization of M cells independently of 62 cells. The centrifuge (Model mustard; PSA, Puck's solution A: CLF, cross-link factor, GSH, reduced glutaJ-21C fitted with a Model .IF 6 elutriator rotor, Beckman Instruments, thione; FBS, fetal bovine serum; MEM, minimum essential medium; SDS, Palo Alto, CA) was operated at a constant rotor speed of 1700 rpm. sodium dodecyl sulfate. 2324 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research. CELL CYCLE DEPENDENCE Between 1 x 10* and 2.5 x 10" cells suspended in 20 ml of medium were introduced into the elutnation chamber at a flow rate of 12 ml/ min and elutriated at room temperature by increasing the flow rate of medium (a-MEM containing 5% FBS and DNase, 20 Mg/ml) through the chamber in a stepwise manner from about 12 to about 30 ml/min. In all, twelve SO-mlfractions were collected and placed on ice. Aliquots were withdrawn for cell count and Coulter volume determinations, and another aliquot was fixed (in 1 ml saline:3 ml 70% ethanol) for analysis by flow cytometry to determine the purity of the individual fractions using an ICP-21 flow cytome ter (Ortho Instruments, Westwood, MA). The proportion of cells of each phase in a particular fraction was determined as described by Johnston et al. (12). Drug Treatments. Either asynchronous cells or cell fractions syn chronized by centrifugal elutriation were sedimented by centrifugation, resuspended in medium (containing 20% FBS) at a density of about 1.3 x 11)"/ml.and plated onto 60-mm tissue culture dishes to give about 2 x 10*cells/dish. After incubation for 30 min at 37*C almost complete attachment of the cells was observed. The cells were then washed twice with PSA and exposed to various concentrations of HN2 in serum-free a-MEM for 0.5 or l h at 37'C. Stock solutions of HN2 (mechlorethamine hydrochloride; Merck, Sharp, and Dohme, West Point, PA) were stored frozen in physiological saline at a concentration of 100 pg/ml. Aliquots were thawed for 0.5 h at room temperature just before use and then diluted to the required concentration with serum-free a-MEM. After treatment, the cells were washed twice with PSA and either trypsinized immediately (0.025% trypsin) for 7 min at 37*C (cells for cytotoxicity or initial DNA damage determination) or were incubated at 37*C in complete McCoy's medium for periods up to 6 h before trypsinization to allow for repair of the H N2-induced DNA cross-links. Cytotoxicity was assessed by colony-forming ability, surviving cells being identified by their ability to form colonies of 50 cells or more. Alkaline Elution. DNA cross-linking was estimated using the alkaline elution methodology originally developed by Kohn (8), with slight modification (6). Briefly, cells were labeled overnight with [MC]thymi- OF DNA CROSS-LINKING linear regression analysis. The error limits associated with the slope values represent the 95% confidence interval. Any DNA strand breaks induced by the drug treatment would be detected as an increased rate of elution in the alkaline elution profiles obtained from 11N2 treated unirradiated control samples that were run simultaneously with the 4 Gy-irradiated samples. Glutathione. GSH was determined as follows. Various numbers (be tween 0 and 10 x IO6) of cells were pelleted by centrifugation (2000 rpm for 10 min at 25*Q, washed by resedimenting from PSA, and then resuspended in 4 ml of distilled water. After vortexing the suspension for 5 min, metaphosphoric acid (1 ml, 25%) was added, and after a further 5 min of vortexing, cell debris was removed by centrifugation (3000 rpm for 20 min) followed by passage of the supernatant through a 0.45-/»mMillex filter (Millipore Corp., Bedford, MA). GSH in the supernatant was estimated using the fluorescent dye 0-phthalicdicarboxaldehyde (15). The relative fluorescence emission was a linear function of the number of cells pelleted within this range but flattened out somewhat at higher cell numbers (data not shown). The fluorescence of the GSH standard solutions was also linear over the range 0-0.05 HIM.GSH concentrations were determined from the relationship GSH = -- x 10-" mol/cell ¿nr where n was the number (in millions) of cells assayed, /was the sample fluorescence, and F was the fluorescence from a standard 0.01 HIM GSH solution. RESULTS Cytotoxicity The cell cycle phase dependence of HN2 cytotoxicity deter mined after exposure of cells from each elutriator fraction to a single dose (3 ng/m\ for 1 h) of HN2 is shown in Fig. 1. Cells in G] were the most sensitive, followed by cells at the GrSphase transition; cells in mid to late S phase tended to be the most resistant. Cells varied in sensitivity by a factor of about dine (0.01 /iCi/ml; 50 mCi/mmol; Schwarz/Mann, Orangeburg, NY) and then chased for 8 h with label-free medium. After drug treatment and trypsinization, an appropriate volume to give 8 x 10s cells was impinged onto a 25-mm polycarbonate membrane (2-pm pore; Nudepore Corp., Pleasanton, CA). The cells were then rinsed twice with icecold PSA containing 5 HIMEDTA, lysed with 5 ml of SDS lysis solution (0.025 M EDTA-2% SDS, final pH 9.7) and then rinsed with 0.1 5 ml of 0.02 M EDTA (pH 10.3). The DNA was eluted overnight in the dark with 0.1 M tetrapropyl ammonium hydroxide-)1.02 M EDTA (free acid)-0.1% SDS (final pH 12.15) at a constant flow rate of 0.04 ml/min to give 10 equal fractions of about 3.5 ml. The DNA in the resulting fractions and that remaining both on the membrane and on the interior of the membrane holder at the completion of the elution experiment was measured by liquid scintillation counting (6). The rate of elution of DNA from HN2-treated cells from the membrane may be influenced by both DNA-interstrand and DNA-protein cross-links (13). Although the use of SDS lysis and elution solutions combined with polycarbonate membranes served to minimize the effect of DNAprotein cross-links on the elution kinetics (14), additional experiments 0.001were performed in which proteinase K (0.5 mg/ml) was added to the lysis solution and retained in contact with the membrane for 0.5 h prior to the elution of the DNA. This treatment further reduces the influence of DNA-linked protein molecules on the elution rate. Those lesions resistant to digestion are presumed to be predominantly DNA-inter strand cross-links, whereas those removed by the proteinase K are 0.0001 presumed to represent DNA-protein cross-links (13, 14). 14 12 10 8 Determination of DNA Cross-Linking. To determine DNA crossFraction Number linking, the cell suspensions were irradiated on ice with 4 Gy of X-rays Fig. 1. Age-response pattern for Chinese hamster ovary cells exposed to a immediately prior to the alkaline elution to introduce a constant num ber of DNA strand breaks (6). CLFs were calculated from the ratio of single concentration (3.0 Mg/ml) of HN2. Cells were separated into fractions enriched in different phases of the cell cycle by centrifugal elutriation and then the log of the fraction of uneluted DNA for the 4-Gy-irradiated control treated with HN2 (l h at 37*C) in medium supplemented with 15% fetal bovine to that for the 4-Gy-irradiated UN2-treated sample, both values being serum. Following treatment, the cells were plated out for colony formation. The determined at the same eluted volume (21 ml in this case, although the early fractions (4-5) represent cells enriched in d (e.g., fraction 4 was 96% d, 3% S phase, 1%G2 + M); middle fractions (6-10) were enriched in S phase (e.g., relative CLFs did not depend greatly on the actual volume selected). fraction 8 was 30% G,, 68% S phase, 2% <;, + M); and later fractions (11-13) The slopes of the resulting cross-linking dose-response curves (ex are enriched in G2 + M (e.g., fraction 12 was 5% G,, 30% S phase, 65% G2 + pressed as change in CLF per 0.1 Mg/ml HN2) were determined by M). Points, average of three separate measurements; hurs. SE. 2325 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research. CELL CYCLE DEPENDENCE OF DNA CROSS-LINKING treatment protocol for the subsequent synchronized cell studies. A treatment time of 0.5 h was used in all of these experiments in an attempt to minimize the repair of lesions during treat ment. Typical alkaline elution profiles from an experiment in which cells were trypsinized immediately after treatment with various concentrations of HN2 are shown in Fig. 4. The elution rate for DNA from HN2-treated cells relative to untreated control cells decreased progressively as the concentration of HN2 was increased between 0-0.6 /¿g/ml,indicating extensive DNA cross-linking. The alkaline elution technique was suffi ciently sensitive that relatively low concentrations of HN2 were required to produce detectable levels of cross-linking. The doseresponse curve relating the initial CLF (zero repair time) to the HN2 concentration calculated from profiles such as those in Fig. 4 is shown in Fig. 5A. The inclusion of proteinase K digestion during the lysis stage decreased the measured cross- 0.03 0.01- 0.003 HN2 Concentration, pg/ml Fig. 2. Dose-response survival curves for CHO cells from elutriator fractions 5 (•)and 10 (O) after exposure to varying concentrations of HN2. Drug treat ments were performed at 37*C for 0.5 h in serum-free medium. Points, average of three separate measurements. Ban, SE. OS "»TA 0.1- 0 40 »1200 40 801200 40 80 120 0.03- CHAME. NUMBt ffWUh«ON* Contint) Fig. 3. Typical histograms from flow cytometric analysis of the DNA content nl (.-I) elutriator fraction 5, (B) elutriator fraction 10, and (Q the unseparated asynchronous population. 36 9 Hours of Button 10 across the cell cycle in response to this particular treatment. Fig. 2 shows complete dose-response survival curves determined for two of the fractions, fractions 5 and 10, which were selected on the basis that they were, respectively, the most sensitive and most resistant fractions when exposed to the single concentra tion of HN2 (Fig. 1). It should be noted that for the experiments in Fig. 2 and in all subsequent data the treatment time was shortened to 0.5 h, which reduced the overall HN2 cytotoxicity, and serum-free a-MEM was used for the drug treatments since preliminary experiments showed that both the cytotoxicity and DNA cross-linking activity of HN2 were modified in a variable manner by the presence of FBS. The shapes of the two survival curves were identical within experimental error. The D0 values (i.e., the dose required to reduce survival by a factor of e~\ calculated from the slope of the exponential part of the survival curve) were 0.64 ng/m\ (fraction 5) and 0.90 Mg/ml (fraction 10), respectively, giving a D0 ratio of 1.4. Fraction 5 was routinely composed of 85-90% GI cells; fraction 10 was usually composed of about 45% S-phase and 55% G2 cells. Typical DNA histograms from flow cytometric analysis of the unsepar ated asynchronous population as well as elutriator fractions 5 and 10 are shown in Fig. 3. The degree of separation achieved for the other elutriator fractions was similar to that reported previously (11). DNA Damage Asynchronous Cells. Preliminary experiments were per formed with asynchronous cells in order to establish a suitable Fig. 4. Typical alkaline elution profiles for DNA from asynchronous Chinese hamster ovary cells. The profiles are representative of the following treatment schedules: (O) no drug treatment, no irradiation; (•)no drug treatment, 4-Gy irradiated; ( ) HN2 (0.2 /ig/ml for O.S h) then 4-Gy irradiated; (T) HN2 (0.4 »ig/mlfor 0.5 h) then 4-Gy irradiated; (•)HN2 (0.6 /ig/ml for 0.5 h) then 4-Gy irradiated. Irradiations were performed in vitro on ice just prior to alkaline elution analysis. HN2 Concentration, ug/ml Time after treatment, h Fig. 5. A, degree of relative DNA cross-linking as a function of HN2 concen tration in asynchronous Chinese hamster ovary cells measured immediately after treatment with HN2 (O.S h at 37*C); B. repair of DNA cross-links in asynchronous Chinese hamster ovary cells treated with HN2 (0.6 ug/ml for O.S h at 37*C). DNA cross-links were measured immediately after and at various times up to 9 h after treatment with HN2. Ban, SE. 2326 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research. CELL CYCLE DEPENDENCE linking by 26 ±4% (SE). When HN2-treated cells (0.6 /tg/ml for 0.5 h) were subsequently incubated in complete medium at 37°Cfor various periods after removal of the drug, the number of cross-links decreased exponentially, with a repair half-time of 4.8 h (Fig. SB). These repair kinetics are similar to those reported for LI210 cells in vitro (16) and for mouse fibrosar coma tumor cells in vivo (17). Proteinase K digestion reduced the cross-linking measured at 6 h after treatment by 28 ±8%, indicating that the two types of lesions (DNA interstrand and DNA-protein cross-links) were repaired with similar kinetics during this time (0-6 h), an observation that we have also previously reported for mouse fibrosarcoma tumor cells In vivo (17). Synchronized Cells. Rather than obtaining limited data for each elutriator fraction, we further characterized the response of the two fractions, 5 and 10, that were the most sensitive and resistant respectively to HN2 (Fig. 1). No DNA strand breaks were detected in cells from either fraction at any time up to 6 h after treatment with HN2 at concentrations up to 0.6 /¿g/ml. It is possible that some low level of strand breakage occurred, but it may have been masked by DNA cross-linking. Extensive DNA cross-linking was observed immediately after the treat ment for both fractions 5 and 10 cells. Dose-response curves relating total initial DNA cross-linking (i.e., DNA interstrand plus DNA-protein) to the HN2 concentration for these fractions are shown in Fig. 6A. Both dose responses were linear over this concentration range, with slopes of 0.65 ±0.11 (fraction 5) and 0.64 ±0.11 (fraction 10), respectively, indicating that the frequency of total DNA cross-link induction was identical in these elutriator fractions at the 95% confidence level. Table 1 shows the results of separate experiments in which cross-linking induced immediately after the treatment was measured either with or without proteinase K digestion. Proteinase K reduced the measured cross-linking by 21 and 32% in fractions 5 and 10, respectively; although these data suggest that there may be a slightly greater proportion of DNA interstrand to DNAprotein cross-links in fraction 5 relative to fraction 10 cells, the actual difference in the frequency of DNA interstrand crosslinking in the two fractions was barely significant at the 95% / 5- -B 4- OF DNA CROSS-LINKING Table 1 Proportion of proteinase K resistant to total DNA cross-linking in elutriator fractions 5 and 10 Cross-linking was measured either immediately after or 6 h after treatment of CHO cells with HN2 (0, 0.2, 0.4, or 0.6 pg/ml; 0.5 h treatment at 37'C). Dose responses were constructed from these data, and the slopes were determined by linear regression analysis. Time (h)0 no.5 total)0.93 Slope" cross-linking (% of ±0.21* With 0.74 ±0.14 0 510 Without 0.91 ±0.25 0 With 0.62 ±0.14 10 0 Without 0.17 ±0.04 6 5 With 0.11 ±0.02 6 510 Without 0.19 ±0.04 6 WithProteinase-resislant 0.13 ±0.0279 6Fraction 10Proteinase-KWithout 68 65 69 'Change in CLF per HN2,0.1 fig/ml. Each slope was determined from at least nine expérimental points using three different drug doses. * Mean ±95% confidence interval. confidence level (Table 1). The capabilities of cells from fractions 5 and 10 to repair cross-links was also compared. Based on the expectation that any differences in repair are probably relatively small, the following procedure was used. Cells were exposed to various doses of HN2 (0-0.6 ng/ml for 0.5 h) and then allowed to repair for 6 h, after which time the cells were harvested and assayed for DNA cross-links using alkaline elution as described above. Complete dose-response curves for DNA cross-linking were then constructed for each of the fractions (Fig. 6.0), and by comparing the slopes of these 6-h dose responses with those determined immediately after treatment (Fig. 6A) we were able to obtain a statistically more reliable estimate of differences in repair rate than could have been achieved from single-dose experiments. The slopes of the 6-h dose-response curves for total DNA cross-linking (i.e., DNA interstrand plus DNAprotein) were 0.19 ±0.03 (fraction 5) and 0.18 ±0.02 (fraction 10), corresponding to the removal of 71 and 72% of the initial DNA cross-links for fractions 5 and 10, respectively; again, these 6-h data showed no significant difference between the two fractions at the 95% level of confidence. As indicated in Table 1, in separate experiments proteinase K digestion reduced the measured cross-linking at 6 h by 35 (fraction 5) and 31% (fraction 10), respectively. Glutathione. Based on reports (18-20) that cellular thiols such as GSH are potentially important moderators of cell sensitivity to alkylating agents such as melphalan and also that nonprotein sulfhydryl levels may be cell phase dependent (21, 22), GSH levels were determined for the synchronized cell populations used in this study. The concentration of GSH in the asynchronous cells was 2.42 ±0.47 nmol/106 cells. GSH levels for the individual elutriator fractions are shown in Table 2 and generally increased through the cell cycle in a manner that could be predicted on the basis of the increase in cell volume; for example, the measured GSH levels in fractions 5 and 10 were 1.54 ±0.41 and 2.66 ±0.58 nmol/106 cells, I I3 § 0.2 0.4 0.6 O 0.2 0.4 respectively. When these values were corrected for the increase in Coulter volume, the GSH levels were identical within exper imental error, the relative normalized values being 1.0 (fraction 5) and 0.94 (fraction 10), confirming a previous report (23) that the intraccllular GSH concentration in CHO cells is relatively constant throughout the cell cycle. 0.6 HN2 Concentration,pg/ml Fig. 6. Degree of relative DNA cross-linking as a function of HN2 concentra tion in Chinese hamster ovary cells synchronized by centrifugal elutriation, measured (A) immediately after or (B) 6 h after treatment with HN2 (0.5 h at 37*Q for cells from elutriator fractions 5 (•)and 10 (O). Points, average of either 10 (0-h data) or 6 (6-h data) separate measurements. Kars, SE. DISCUSSION Establishing the relationships between DNA damage, the repair of this damage, and the cytotoxic effects of antitumor 2327 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research. CELL CYCLE DEPENDENCE Table 2 Variation in GSH levels through the CHO cell cycle Cells were synchronized by centrifugal elutriation, and the GSH in these synchronized populations was determined by a modification of the procedure described by Cohn and Lyle (15). Elutriator fraction cells)34567.95 GSH concentration (nmol/ 10* 0.46°.66 ± 0.428 0.409 0.3810 0.5811 ±0.5112 0.35.54 ± 0.41.62 ± 0.35.93 ± ± 2.03 ± 2.12 + 2.66 ± 2.76 3.49 ±0.60 * Mean ±SE of four separate measurements. agents is an important objective in understanding the mecha nism of action of such drugs. The cell cycle specificity exhibited by these agents provides an interesting situation in which to address some of these basic questions. The data shown in Fig. 1 indicate that cells in (•,are the most sensitive to HN2, in agreement with the results of previous studies relating to alkylating agents in general (2) and also with data specifically relating to HN2 (24-26). Although the alkylation of DNA by HN2 results in a variety of different lesions, diadducts such as DNA inter-strand cross-links are presumed to be particularly cytotoxic (8), based on the fact that this bifunctional drug is considerably more cytotoxic than its monofunctional counter part (27). Perhaps the best evidence relating DNA damage to cytotoxicity is that the cytotoxic effects can be modified by DNA repair (e.g., see Ref. 28), also suggesting that repair processes themselves represent an important mechanism of moderation of drug action; thus, any differences in the initial level of damage and/or in the subsequent response of the cell to that damage (i.e., in repair capacity) through the cell cycle are probably important factors in determining the ultimate pattern of cytotoxicity. In previous studies of this type, survival has usually been determined after exposure of synchronized cells to a single drug concentration, although considerably more information may be obtained when complete dose-response curves are generated for each cell cycle phase (29). In initial experiments, therefore, surviving fraction was determined over a range of drug concen trations (Fig. 2) for the two elutriator fractions (5 and 10) that showed the greatest differential sensitivity to killing by HN2. This analysis revealed an important point in that although the variation in surviving fraction through the cell cycle in response to a single dose of HN2 was as great as a factor of 10 when the treatment resulted in low survival levels (Fig. 1), this change was in fact accounted for by a ratio of A> values of only 1.4. Although this situation is simply a consequence of the divergent survival curves, it is the change in survival curve parameters reflected in the ratio of terminal slopes (i.e., of A> values) that indicates differences in intrinsic cell sensitivity. The first question that we addressed was whether there were measurable variations in the initial yield of DNA damage in cells treated with HN2 in different phases of the cell cycle. It is clear from the data shown in Fig. 6A that there was no signifi cant difference in total (DNA interstrand plus DNA-protein) DNA cross-linking between elutriator fractions 5 and 10 im mediately after the drug treatment; furthermore, the use of proteinase K digestion to resolve any possible difference in the contribution of DNA interstrand and DNA-protein cross-links to the total cross-linking effect (Table 1) revealed that the relative frequency of DNA interstrand cross-link induction in OF DNA CROSS-LINKING both fractions S and 10 cells was also indistinguishable at the 95% level of confidence. The observed 1.4-fold difference in cytotoxicity cannot therefore be readily explained in terms of alterations in initial cross-linking levels. We therefore determined whether cells from the two fractions differed in their capacity to remove these DNA cross-links; however, cells from both fractions 5 and 10 appeared to be equally efficient in the removal of cross-links (Fig. 6) in the first 6 h after treatment. Proteinase K digestion again revealed that the proportion of DNA interstrand to DNA-protein cross links was identical within experimental error in each fraction (Table 1), indicating that there was no differential repair of one or the other type of lesion in the different phases; furthermore, the fact that the proportion of DNA interstrand to DNAprotein cross-links measured after 6 h was identical to that measured immediately after the drug treatment indicates that both types of lesions (DNA interstrand and DNA-protein cross links) were repaired with similar kinetics during this time; however, the possibility of subtle changes that cannot be re solved within the limitations of available DNA damage assays cannot be completely ruled out. These observations agree with a previous study (26) which also suggested that neither the frequency nor repair of DNA interstrand cross-links varied among CHO cells that were synchronized into the different cell cycle phases and treated with HN2, even though the earlier study (26) used a less sensitive assay for DNA cross-linking, which required a much higher concentration of HN2 (approxi mately, 6 ng/ml), and would have given a surviving fraction of less than Id4. Because of the increased sensitivity of the alkaline elution assay, DNA damage in the present study could be determined at concentrations for which the degree of cell killing was much lower (never greater than 50%). It should also be noted that in a study of this type where the loss of detection of the DNA cross-link is the measured end point, the possibility remains that differences in the efficiency of subsequent repair steps could still account for the observed differences in cytotox icity. In conclusion, we confirm the general finding that cells in ( ¡, are more sensitive to HN2 than those in late S phase or G2. This sensitivity is apparently a result neither of variations in initial DNA cross-linking (either DNA interstrand or DNAprotein) nor of differences in DNA cross-link repair capacity in the different cell cycle phases; on the other hand, considering that the repair half-times measured here are on the order of 4 h, it seems reasonable to hypothesize that the sensitivity of cells in the different cell cycle phases could be related to the proba bility of repairing these lesions prior to the onset of DNA synthesis, as suggested by Ludlum (30). In such a model, cells treated in G2 would obviously have a survival advantage over those treated at the d-S-phase transition. ACKNOWLEDGMENTS We thank Raul Laurel for excellent technical assistance. REFERENCES 1. Walker, I. G., and Helleiner, C. W. 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Meyn Cancer Res 1986;46:2324-2329. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/46/5/2324 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.