Topoisomerase activity in irradiated mammalian cells

Mutation Research, 216 (1989) 43-55 Elsevier

43

MTR 08712

Topoisomerase activity in irradiated mammalian cells Raymond

L. W a r t e r s , B r a d l e y W . L y o n s , K a t h r y n K e n n e d y a n d T. M u a L i

Department of Radiology, University of Utah Health Sciences Center, Salt Lake City, UT 84132 (U.S.A.) (Received 20 June 1988) (Revision received 12 September 1988) (Accepted 20 September 1988)

Keywords: Topoisomerase II; Double-strand break repair; Radiation

Summary The role of topoisomerase enzymes in the response of HeLa S3 cells to ionizing radiation was investigated. Exposure of cells to 100 Gy of X-radiation had no detectable effect either on the total cellular topoisomerase activity as measured by the relaxation of supercoiled plasmid DNA by cell sonicates or on the total cellular topoisomerase II activity as measured by plasmid DNA catenation. Total topoisomerase II activity remained constant for up to 90 rain after cell irradiation. The effect of 2 drugs (caffeine and novobiocin) which inhibit topoisomerase II activity on the HeLa cell response to radiation was determined. Both drugs were found to inhibit topoisomerase II in vitro and to inhibit the recovery of nucleoid sedimentation in irradiated cells in vivo to the same extent. Topoisomerase II was inhibited by 50% by exposure to 10 mM caffeine and 0.79 mM novobiocin. At low concentrations neither drug affected the induction frequency, nor the rejoining rate, of DNA double-strand breaks. Caffeine (5 mM) inhibited the short-term recovery of cells from radiation while novobiocin (0.79 mM) had no detectable effect on the capacity of cells to recover from radiation exposure. The results indicate that topoisomerase II is not required for DNA double-strand break rejoining though it could be required for the recovery of DNA coiling in the irradiated cell. If topoisomerase II is involved at all in cell recovery from irradiation, this role does not apparently involve an ATP-dependent enzyme activity.

Exposure to low concentrations of the drug novobiocin (200 t~g/ml or 0.32 mM), a type-II topoisomerase (topo) inhibitor, has been shown not to inhibit the repair of ionizing radiation-induced DNA damage, measured either as the removal of DNA single-strand breaks from irradiated cells (Collins and Johnson, 1979), or as an increase in the frequency of radiation-induced sis-

Correspondence: Raymond L. Warters, Ph.D., Department of Radiology, University of Utah Health Sciences Center, Salt Lake City, UT 84132 (U.S.A.).

ter-chromatid exchanges (Morgan et al., 1986). In addition, low concentrations of novobiocin have not been found to affect cellular responses to ionizing radiation measured as long-term cell survival (Collins and Johnson, 1979; Mattern and Scudiero, 1981). While these studies indicated that topoisomerase II plays no direct role in the cell's response to ionizing radiation, these studied did not demonstrate that the drug concentrations used actually affected significantly the endogenous cellular topoisomerase II activity. Since topoisomerase II enzymes may be significantly more resistant to the drug novobiocin in mammalian

0165-1161/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

44

cells, higher drug concentrations than have been used previously may be required to observe druginduced changes in the cell's response to ionizing radiation. Using an assay which measures the ability of cellular topoisomerase enzymes to alter the topological state of plasmid DNA, the present study attempted to determine the effect of ionizing radiation exposure on the endogenous cellular activity of the mammalian topoisomerase enzymes. The influence of novobiocin, at concentrations demonstrated to inhibit the majority of topoisomerase II enzyme activity, on the repair of radiation-induced D N A damage, and on the single cell survival of irradiated cells, was measured. Material and methods

Cell culture and labeling HeLa S3 cells (doubling time ca. 24 h) were maintained in exponential growth in monolayer culture with McCoy's 5A medium supplemented with 5% fetal bovine and 5% calf serum. Monolayer cultures of HeLa cells were uniformly labeled in their D N A or protein by exposure for 18 24 h to whole medium containing [Me-14C]thymidine at 0.07/~Ci/ml or an equal mixture of [3H]leucine and [3H]lysine at 0.25/~Ci/ml. For pulse-labeling studies, [14C]thymidine-prelabeled cells were exposed to 0.2 /~Ci/ml of either [3H]thymidine or [3H]uridine for 30 rain at 37°C. Cells were exposed to X-irradiation with a Philips model RT 250 X-ray unit at 250 kVp, 15 mA, with 3 mm of aluminum filtration at a dose rate of 2.5 G y / m i n . Cell survival was determined by the cloning efficiencies of single cells as previously described (Warters and Henle, 1982).

DNA damage assays D N A single-strand break induction was measured by the alkaline (pH 12.2) filter elution assay as previously described (Warters et al., 1985b). In all the experiments discussed the D N A radioactivity elution kinetics were observed to be first-order with respect to elution volume regardless of treatment (X-irradiation or drug exposure). Since the D N A elution rate from polycarbonate filters at pH 12.2 has been shown to be a function of the molecular weight of single-stranded D N A (Kohn

et al., 1976; Warters et al., 1987) we calculated an elution rate or single-strand scission factor (SSSF) as described by Murray et al. (1984) using the relationship:

F~

SSSF = - l o g l 0 ~

where F was the fraction of total D N A remaining on the polycarbonate filter after a total of 15 ml of elution with pH 12.2 buffer in treated (T) or control (C) cells. The neutral filter elution technique used in these studies to measure D N A double-strand breaks was a modification of the method of Bradley and Kohn (1979) as previously described (Warters et al., 1985a). In short, between 2 and 5 × 105 total cells in ice-cold phosphate-buffered saline were collected onto 25-mm diameter, 1-/zm pore-size, polycarbonate filters. The cells were lysed for 60 min at room temperature with 3 ml of lysis solution (2% SDS, 0.025 M N a z E D T A , 0.05 M glycine, 0.05 M Tris, pH 7.2) containing 1 mg of proteinase K. After lysis, 23 ml of neutral eluting solution (0.05 M glycine, 0.025 M N a 2 E D T A , 2% SDS and 0.05 M Tris, pH 7.2) was added above the filters and the elution solution was pumped across the filters at a rate of 0.025 ml/min. When the eluting solution had been fully collected, the radioactivity contained on the filter, in sixteen 1.5-ml fractions, and in a 10-ml wash (0.5 N NaOH) of the apparatus was determined by scintillation counting. In this neutral filter elution assay the elution kinetics of D N A radioactivity were biphasic with respect to elution volume (results not shown). The relationship described by a plot of D N A damage induction at pH 7.2 versus radiation dose was qualitatively similar regardless of whether the data was expressed as the initial rate of D N A elution calculated in a manner similar to that described above or as the fraction of D N A remaining on the polycarbonate filter after recovery of all elution solution. Since the rate of elution of D N A from the filters at pH 7.2 has not been demonstrated unambiguously to reflect the molecular weight of double-stranded DNA, we expressed our pH 7.2 filter elution data as the fraction of D N A remaining on the polycarbonate filter at the completion of pH 7.2 buffer elution.

45 Nucleoid sedimentation studies The nucleoid sedimentation studies were performed in a manner similar to that of Cook et al. (1976) as previously described (Warters et al., 1985a). Prelabeled cells were washed once with phosphate-buffered saline. Approximately 2 x 105 total cells in 0.2 ml of phosphate-buffered saline were layered onto 0.9 ml of a lysis solution (10 mM EDTA, 1.95 M NaC1, 0.5% Triton X-100 and 2 mM Tris-HC1, pH 8.0) which has been layered over a 36-ml 15-30% sucrose gradient containing 10 mM EDTA, 1.95 M NaC1 and 2 mM Tris-HC1, pH 8.0. The cells were allowed to lyse for 30 min in the dark at ambient temperature. The gradients were centrifuged (SW 28 rotor) at 10000 rpm for 60 min (w2t = 4 rad2/sec x 109) in a Beckman L5-50B preparative ultracentrifuge. Approximately twenty-five 1.5-ml fractions, and a wash of the bottom of the gradient tube, were collected onto glass fiber filters by TCA precipitation and quantitated by scintillation counting. Topoisomerase assays Supercoiled plasmid pBR322 was grown in, and purified from E. coli HB 101 by a modification of the procedure of Clewell and Helsinki (1979) as previously described (Sandhu et al., 1985). HeLa cells, or subcellular fractions, were suspended in a reaction buffer (35 mM Tris-HCl, pH 7.9, 20 mM KC1, 20 mM MgCI z, 10 mM /3-mercaptoethanol, 0.1 mM EDTA, 30 /~g/ml of bovine serum albumin, 2 mM spermidine, and 1.0 mM ATP) and ruptured at 4 ° C with a 15-sec burst of a Heat Systems-Ultrasonics, Inc. model W185F cell sonicator. Sonication conditions were chosen to provide maximum yield to topoisomerase activity from sonicated whole cells. The standard topoisomerase catenation reaction (18 /~1) contained 8 /~1 of a serial dilution with reaction buffer of the cell sonicate, 5 /zl of the reaction buffer and 5 btl of plasmid D N A (0.3 ktg total DNA) in reaction buffer. The reaction mixture was placed at 37 ° C for 60 rain. In some experiments the decatenation activity of enriched HeLa topo II was measured using kinetoplast D N A as a substrate. Kinetoplast DNA networks were isolated from the trypanosome Crithidia fascieulata cultured in Brain Heart Infusion medium (Difco) supplemented with 10 btg/ml

hemin (Sigma). The kinetoplast D N A was prepared from exponential phase cultures and purified on cesium chloride-ethidium bromide density gradients as described by Simpson and Simpson (1974). Topoisomerase II was extracted from HeLa cell nuclei with 1.5 M NaC1, 4% PEG 4000 (Sigma) and enriched by passage through a hydroxylapatite column as described by Miller et al. (1981). Topoisomerase II activity (i.e., an ATP-dependent, novobiocin-sensitive, decatenation activity) eluted principally at 0.5 M potassium phosphate. Fractions containing topo II were dialyzed for 24 h into a buffer containing 10% glycerol, 40 mM Tris-HCl, pH 7.5, 10 mM /3-mercaptoethanol, 1 mM PMSF. A standard decatenation reaction (28 /~1) contained 1-5 ~1 of the enriched topo II, 21-25 /~1 of a reaction buffer (50 mM Tris-HCl, pH 8.0, 120 mM KC1, 190 mM MgC12, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30/~g/ml BSA and 0.5 mM ATP) and 2 #1 of kinetoplast DNA (0.5 /~g DNA total). The reaction mixture was placed at 37°C for up to 60 min. The topological states of the plasmid or kinetoplast D N A were assessed by horizontal electrophoresis in a 1% agarose gel. The gels were subjected to 1.5 V / c m for 18 h in 'E' buffer (30 mM Tris-HC1, 12 mM sodium acetate, 2 mM sodium EDTA, pH 8.0). After electrophoresis, the gels were stained with 100/~g of EtBr in 200 ml of E buffer for 15 min. The gels were then illuminated with a 254-nm light source and photographed. The photographic negatives were used for the quantitation of each electrophoretic band by scanning densitometry with a Gilford model 2600 scanning spectrophotorneter. Results

To determine the potential role of topoisomerase (topo) enzymes in the response of mammalian cells to ionizing radiation, we felt it was necessary first to determine the effect of cell irradiation on endogenous topo activities. To do this we measured the capacity of sonicates, or subcellular fractions, of HeLa cells to change the topological state of plasmid pBR322 DNA. Plasmid DNA isolated from Escherichia coli routinely was greater than 85% closed, circular, supercoiled DNA as assayed by 1% agarose gel electrophoresis (Fig. 1, insert). In the presence of small numbers

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Fig. 1. Topoisomerase catenation activity in HeLa cells. HeLa cells were sonicated and added to a reaction mixture containing plasmid pBR322 DNA, as described in the Methods section. The reaction mixture was placed at 37 °C for 60 min and then electrophoresed through 1% agarose gels. The gel was stained with ethidium bromide, photographed and analyzed by scanning densitometry. Plotted is the fraction of plasmid DNA in either the supercoiled ([3) or catenated (O) state versus the total cell equivalents used in the reaction. Plotted is the average + S.D. from 3 or more repeat Expts. Insert: Photograph of an ethidium bromide-stained gel demonstrating supercoiled (lane a), relaxed (lane b) and catenated (lane c) DNA forms.

of HeLa cell equivalents, supercoiled plasmid D N A was efficiently converted to the relaxed form. O n the average 3 × 10 2 total cell equivalents were sufficient for half conversion of 0.3 /~g of supercoiled plasmid D N A to the relaxed form within 60 m i n at 37 ° C. In contrast, much larger n u m b e r s of cell equivalents (0.9 1.0 × 10 4) were required for 50% conversion of the supercoiled plasmid D N A to a catenated form u n d e r the same c o n d i t i o n s (Fig. 1). Since either the type I or II topoisomerase can relax supercoiled plasmid D N A we assumed that this assay measured both topo I a n d II enzyme activities, while c a t e n a t i o n of plasmid D N A , which requires a D N A d o u b l e - s t r a n d cut, measured only topo II. Consistent with this interpretation we found that the c a t e n a t i o n activity: had an absolute r e q u i r e m e n t for exogenously added A T P ; was inhibited by the a d d i t i o n of the drug novobiocin; a n d proceeded at a b o u t twice the rate in the presence of a n exogenously added p o l y a n i o n such as spermidine. The relaxation activity was unaffected by the presence of absence of A T P or

novobiocin. Thus we assumed that the c a t e n a t i o n assay measured only a topo II-type e n z y m e activity. Since a p p r o x i m a t e l y 33 times more cellular material was required for 50% catenation, than for 50% relaxation, of plasmid D N A we assumed that the topo II-type enzyme activity was 3% or less of the total cellular topo enzyme activity. Thus the relaxation of plasmid D N A could be a s s u m e d to measure principally topo l-type enzyme. W h e n HeLa cells were exposed to increasing doses of X - r a d i a t i o n a n d these two assays performed, we f o u n d that doses of X - r a d i a t i o n up to 100 G y had no a p p a r e n t effect on the total cellular activity of either topo I or II (Fig. 2). Larger radiation doses were not used since single cell survival would be u n d e t e c t a b l e at the highest dose used in this study. While the absence of an exogenously added p o l y a n i o n (i.e., spermidine) reduced

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Fig. 2. Topoisomerase activity in irradiated cells. HeLa cells were exposed to increasing doses of X-radiation, sonicated and added to plasmid DNA for the reaction described in Fig. 1. The number of cell equivalents required for 50% relaxation of supercoiled DNA (E]) or for 50% conversion of plasmid DNA to the catenated form (o) was determined and plotted versus radiation dose. The average_+ 1 S.D. in 3 or more repeat Expts. is plotted in this and subsequent figures. Single points are the average of 2 repeat Expts.

47

the rate of control cell D N A catenation, the absence of spermidine in the reaction mixture did not change the insensitivity, relative to control, of topo II to 100 G y of X-radiation. Thus within a range of radiation doses where some cell survival was observed, radiation exposure had no detectable effect on either topo I- or topo lI-type enzyme activities. In a separate series of 3 experiments H e L a cells were exposed to 100 G y of X-radiation and placed at 3 7 ° C to allow cellular repair. The total cellular activity (average + 1 S.D.) of topo II, relative to the untreated control cells as assayed by the catenation reaction, was 0.864 + 0.240, 0.863 + 0.237 and 0.860 + 0.149, respectively, of the activity observed in the control cells 0, 10 and 90 min after irradiation. Thus while there may have been some radiation-induced inactivation of cellular topo II activity, the total cellular activity appeared to remain constant after irradiation. Since radiation exposure had no direct effect on either topoisomerase ! or II activity, we determined the effect of the topoisomerase II inhibitor novobiocin on the radiation response of H e L a cells. N o v o b i o c i n is a nonspecific inhibitor of topoisomerase II, and inhibits a n u m b e r of ATPrequiring enzymatic reactions in vitro, including those of topo II. While specific conclusions regarding topo II function cannot be drawn directly from results from novobiocin-exposed cells, high intracellular drug concentrations are likely to impair endogenous topo II activities. As previously reported (Mattern and Scudiero, 1981), novobiocin exposure inhibited pulse incorporation of both [3H]thymidine and [3H]uridine into H e L a cells (Fig. 3A). In cells exposed to 0.79 m M novobiocin, m a x i m u m inhibition of pulse incorporation of [3H]uridine, or [3H]thymidine, occurred with a half-time of 10 and 30 min, respectively. Once m a x i m u m drug inhibition of "precursor incorporation was reached (by 30 and 60 min for [3 H]uridine and [3H]thymidine, respectively), it remained at a constant level for the duration of drug exposure. [3H]Thymidine incorporation was considerably more sensitive (50% inhibition at 0.32 m M ) than was [3H]uridine incorporation (50% inhibition at 0.79 m M ) to novobiocin exposure (Fig. 3A). W h e n cells were exposed to increasing concentrations of novobiocin and the capacity of cell sonicates to catenate plasmid D N A in the presence of the

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0.1 Fig. 3. Drug-induced inhibition of topoisomerase II activity. HeLa cells were exposed to increasing concentrations of novobiocin (A) or caffeine (B) as indicated for 60 min at 37° C. The cells were then pulse-labeled with [3H]thymidine (t3) or [3H]uridine (I) for 30 rain, and TCA-precipitable radioactivity was determined. Fractional incorporation of radioactivity into DNA or RNA was expressed as the ratio of the total 3H radioactivity incorporated into drug-treated cells versus the total 3H radioactivity incorporated into control cells. Alternately topoisomerase activity was determined either as the fractional reduction in DNA strand break induction by mAMSA (zx) as described in Fig. 5, or the capacity of cell sonicates to catenate plasmid DNA (0).

same drug concentration was measured, we found that this in vitro topo II activity was inhibited with a kinetic most similar to that observed for inhibition of R N A synthesis (Fig. 3A). W h e n cells were exposed to increasing concentrations of caffeine for 60 rain and then assayed, in a similar manner, for either [3H]thymidine incorporation (Fig. 3B) or plasmid catenation (results not shown) very similar inhibition kinetics were observed for both processes (Fig. 3B). [3H]Thymidine pulse incorporation and plasmid catenation were inhibited by 50% after exposure to 12 and 15 m M caffeine, respectively. A n o t h e r method for determining topoisomerase II activity is to measure D N A strand breakage induced by topoisomerase II in cells exposed to the intercalating drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). This interaction can be illustrated by the inhibition of H e L a cell topo II decatenation of kinetoplast D N A (Fig. 4). The kinetoplast D N A network is too large to enter a 1% agarose gel (Fig. 4, lane 2). Decatena-

48

tion of kinetoplast D N A by HeLa cell topo II results in liberation of 2500-bp D N A circles which do enter the gel (Fig. 4, lane 3) and coelectrophorese with supercoiled plasmid D N A . This topo II decatenation activity requires ATP (Fig. 4, lane 5) and is inhibited in the presence of novobiocin (Fig, 4, lane 6). Exposure to increasing concentrations of m - A M S A progressively inhibits this decatenation activity (Fig. 4, lanes 7-10). Since mA M S A inhibits the D N A strand passage activity of topo II, exposure of intact HeLa cells to this drug results in topoisomerase II-linked D N A strand breaks with topo II crosslinked through a tyrosine residue to the 5' terminus of the D N A (Minford et al., 1986; Nelson et al., 1984). In the intact cell this interaction is detected as a D N A strand break, or reduction in D N A size, if D N A is treated with a protein denaturant such as proteinase K (Zwelling et al., 1981). When HeLa cells were exposed to increasing concentrations of mAMSA and their D N A size assayed by pH 12.2

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Fig. 4. Inhibition of kinetoplast decatenation by m-AMSA. Topoisomerase I1 was enriched from HeLa cells and kinetoplast D N A networks from Crithidia as described in the Methods. Untreated k D N A (lane 2), or k D N A treated with HeLa cell topo II in the presence (lane 3) or absence (lane 5) of ATP, or in the presence of 15 m M caffeine (lane 4), 0.79 m M novobiocin (lane 6) or increasing concentrations (as indicated) of m-AMSA (lanes 7-10) was placed at 37 ° C for 30 min. The topological state of k D N A was measured by electrophoresis through 1% agarose gels as described in Fig. 1. Supercoiled plasmid pBR322 D N A (lane 1) was included for reference.

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Fig. 5. Intercalator-induced D N A strand breakage in HeLa cells. HeLa cells were exposed to increasing concentrations (/~M) of the intercalating drug m-AMSA (as indicated) at 37 o C for 30 min. The cells were collected by trypsinization and their D N A analyzed by alkaline filter elution as described in the Methods section. Plotted in panel A is the fraction of D N A radioactivity remaining on polycarbonate filters during pH 12.2 elution in cells exposed to various concentrations of m-AMSA (as indicated in /zM), A DNA single-strand scission factor (calculated as described in the Methods section) is plotted in panel B versus drug exposure concentration.

49

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Fig. 6. DNA size distribution in m-AMSA-exposedcells. (A) HeLa cells were exposed to 1.0 (D), 10.0 (11)or 50.0 (o) ~M m-AMSA for 30 rain at 37 °C and their DNA analyzed by sedimentation through alkaline sucrose gradients (total w2t = 5.1 rad 2 sec × 101°). The distribution of DNA from cells exposed to 25 and 50 ~M m-AMSA was the same. (B) Cells were either exposed to 50 ~M M-AMSA for 30 rain (o), irradiated with 10 Gy of X-radiation and then exposed to 50 #M m-AMSA at 37°C for 30 min (O) or pulse-labeled with [3H]thymidine for 30 min (D) at 37 o C. Cell DNA was then analyzed by sedimentation through alkaline sucrose gradients (total w2t = 8.2 rad 2 sec × 101°). More than 90% of 14C-labeled, parental DNA radioactivity in unirradiated cells or cells exposed to 10 Gy of X-radiation only sedimented beyond fraction 17 or to the bottom of the gradients. Direction of sedimentation is from left to right.

filter elution in the presence of proteinase K, a drug c o n c e n t r a t i o n - d e p e n d e n t increase in D N A s t r a n d breakage was observed (Fig. 5). The e l u t i o n kinetics of D N A from cells exposed to 1.0 /~M m - A M S A was quite similar to that observed in cells exposed to 10 G y of X - r a d i a t i o n (results not shown). Since little, or no, elution of D N A at p H 7.2 was observed at this drug c o n c e n t r a t i o n we assumed that exposure to m - A M S A p r o d u c e d a p r e p o n d e r a n c e of D N A single-strand breaks. W h e n D N A from cells exposed to 1.0 /~M mA M S A was analyzed by alkaline sucrose gradient s e d i m e n t a t i o n , the D N A size was a b o u t 1 5 0 - 1 6 0 S, i n d i c a t i n g a p p r o x i m a t e l y 1 strand break per 5 0 0 - 1 0 0 0 kb (Fig. 6A). Exposure to drug concentrations greater than 1.0 /~M c o n t i n u e d to reduce the D N A size (Fig. 6A) b u t a p p a r e n t l y in a

n o n u n i f o r m fashion up to a c o n c e n t r a t i o n of 25 /tM at which n o further D N A s t r a n d breakage occurred (Fig. 6A). T h e D N A size at or above this drug c o n c e n t r a t i o n was a p p r o x i m a t e l y that observed for a H e L a cell replicon (Fig. 6B) indicating that the frequency of active topo II in these HeLa cells is a b o u t 1 enzyme molecule per replicon (ca. 60 kb). If H e L a cells were exposed to 10 G y of X - r a d i a t i o n a n d then to 50 ttM m - A M S A for 30 m i n at 3 7 ° C a n d their D N A analyzed by alkaline sucrose gradient s e d i m e n t a t i o n (Fig. 6B) a D N A size d i s t r i b u t i o n identical to the one observed in cells exposed to 50 /~M m - A M S A o n l y was observed. C o n s i s t e n t with the results o b t a i n e d in the in vitro assay (Fig. 2), this result indicated that r a d i a t i o n exposure does n o t affect detectably the capacity of topo II to f u n c t i o n in the cell.

50

When HeLa cells were exposed to increasing concentrations of novobiocin (Fig. 3A) or caffeine (Fig. 3B) for 60 min before exposure to 1.0 /LM m-AMSA, somewhat different results were obtained. In contrast to a previous report (Pommier et al., 1984), novobiocin was effective at reducing m-AMSA-induced D N A strand breaks (i.e., mAMSA-induced topo II-linked D N A breaks). This was consistent with a partial requirement for ATP for this interaction to occur in vivo. In contrast, 15 mM caffeine was as efficient (ca. 50%) at reducing this topo II m-AMSA interaction as it was in inhibiting D N A synthesis or in inhibiting kinetoplast D N A decatenation in vitro by enriched HeLa topo II (Fig. 4, lane 4). One D N A 'repair' process previously demonstrated to be inhibited by exposure to novobiocin is the reconstitution of the sedimentation properties (DNA supercoiling) of the 'nucleoid' in irradiated cells (Cook and Brazell, 1975; Mattern et al., 1983). In HeLa cells, exposure to 10 Gy of X-radiation resulted in a reduction in the nucleoid sedimentation rate (Fig. 7A). This phenomenon has been ascribed to the production by ionizing radiation of strand breaks in nuclear D N A causing the

relaxation of negative D N A supercoils (Cook and Brazell, 1975; Bryant et al., 1984). When 10 Gyirradiated cells were placed at 3 7 ° C to allow D N A damage repair, nucleoid sedimentation recovered to control rates with a half-time of 60 min (Fig. 7A and B). While exposure to 0.79 mM novobiocin for up to 5 h had no effect on the sedimentation of either the unirradiated or irradiated nucleoid (results not shown), reconstitution of control level nucleoid sedimentation in irradiated cells during exposure to 0.79 mM novobiocin occurred with a half-time of 150 min (i.e., was inhibited by a factor of 0.6) (Fig. 7B). Thus at novobiocin concentrations up to 0.79 raM, both in vitro plasmid D N A catenation by topoisomerase II (Fig. 3A) and in vivo nucleoid reconstitution had the same apparent sensitivity to novobiocin exposure. When HeLa cells were exposed to higher novobiocin concentrations (1.1-1.6 mM) the sedimentation distance of the nucleoid from unirradiated cells increased with increasing drug exposure time. So no radiation studies were performed in this drug concentration range. Exposure to 5-30 mM caffeine for 30-120 min had no detectable effect on the sedimentation behavior of the HeLa

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Fig. 7. Sedimentation of nucleoids from irradiated and drug-treated HeLa cells in neutral sucrose gradients. (A) Unirradiated cells (zx) or cells exposed to 10 Gy of X-radiation and placed at 37 ° C for 0 (A), 30 ( o ) and 90 ( I ) min were lysed above and sedimented through neutral sucrose gradients as described in the Methods section. Direction of sedimentation is from left to right. (B) Fractional recovery of the unirradiated nucleoid sedimentation rate in control cells ( o ) or cells incubated in the presence of either 0.79 mM novobiocin ( I ) or 5 mM caffeine (e) for 60 min before irradiation and during post-irradiation recovery.

51

cell nucleoid (results not shown). However in cells previously exposed to 5 or 10 m M caffeine for 60 min, reconstitution of control level, nucleoid sedimentation subsequent to a 10 G y exposure to X-radiation occurred with a half-time of 102 (Fig. 7B) or 180 rain (results no shown) respectively, and thus was inhibited by a factor of 0.41 and 0.67, respectively. Exposure of H e L a cells to 0.79 or 1.58 m M novobiocin, or 5 m M caffeine, produced no detectable D N A single- or double-strand breakage. When H e L a cells were exposed to 0.79 or 1.58 m M novobiocin, or 5 m M caffeine, for 60 min prior to irradiation no enhancement was observed in the induction by radiation of either D N A single-strand breaks as assayed by the alkaline (pH 12.2) filter elution assay (data not shown) or

of D N A double-strand breaks as assayed by the p H 7.2 filter elution assay (Fig. 8A). Similar findings have been reported previously for D N A single-strand break induction in cells exposed to novobiocin (Collins and Johnson, 1979) or caffeine (Lehman, 1972). The initial rate of rejoining of D N A single-strand breaks (T1/: = 5 min) following exposure to 10 Gy of X-radiation was the same in both control cells and cells previously exposed for 60 min to either 0.79 m M novobiocin or 5 m M caffeine (results not shown). The initial rate of removal of D N A double-strand breaks (7"1/2 = 15 min) following exposure to 80 G y of X-radiation was the same in both control cells and in cells exposed for 60 min to 0.79 m M novobiocin, while pre-exposure to 1.58 m M novobiocin increased the half-time (T1/2 = 30 min) for re-

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Fig. 8. Radiation-induced DNA double-strand break induction and rejoining in drug-treated HeLa cells. (A) Cells incubated for 60 rain in 0.79 mM novobiocin (D), 1.58 mM novobiocin (11) or left unexposed ( o ) were exposed to increasing radiation doses and analyzed by pH 7.2 filter elution. Plotted is the fraction of DNA radioactivity remaining on the filter after elution with 25 ml of pH 7.2 buffer. (B) Cells exposed to drugs for 60 rain were exposed to 80 Gy of X-radiation and placed at 37 ° C. Plotted is the fraction of D N A damage (pH 7.2 filter elutability) remaining at the indicated times. Symbols are the same as in A.

52

moval of D N A double-strand breaks (Fig. 8B). As has been reported elsewhere (Weibezahn and Coquerelle, 1986; Rowley and Kort, 1988) exposure to 5 mM caffeine for 60 min before and after 80 Gy X-irradiation had no detectable effect on the initial rate of D N A double-strand break rejoining (results not shown). Thus, while novobiocin and caffeine exposure had no detectable effect on the induction frequency of D N A strand breaks, novobiocin concentrations sufficiently high to inhibit 90% of topo II activity also inhibited D N A double-strand break rejoining. Exposure to 5 mM caffeine for up to 5 h had no detectable effect on cell-cloning efficiency, while exposure to 0.79 mM novobiocin for the same time period reduced survival by 20%. An exposure, time-dependent decrease in cell-plating efficiency was observed in cells exposed to 1.58 mM novobiocin with a survival of 10% after 5 h. When HeLa cells were exposed to increasing doses of radiation (Fig. 9) cell survival decreased with a D 0 of 1.3 Gy. Exposure to 0.79 mM novobiocin for 60 rain before, and for 4 h after, exposure to increasing radiation doses had no apparent effect on the slope of the survival-response curve. Exposure to 5 mM caffeine for 60 min before, and for 4 h after, exposure to increasing radiation doses had no apparent effect on the slope of the response curve (D o = 1.1 Gy). This depression by caffeine of cell survival could be explained as an inhibition in the development of sublethal damage repair (SLDR). When a total dose of 10 Gy was split and delivered in 2 equivalent doses separated by increasing time (Fig. 9, insert) some recovery from radiation cytotoxicity was observed between the 2 doses. Exposure to 0.79 mM novobiocin for 60 min before delivery of the first dose, and between the delivery of the first and second dose, had no affect on SLDR development. In both control and novobiocin-exposed cells, SLDR developed with a half-time of about 90 rain and reached a maximum survival increase of 1.6 (Fig. 9, insert). Continuous exposure to caffeine during a similar treatment significantly inhibited the development of SLDR. Another form of early cellular recovery from radiation-induced cytotoxicity is termed potentially lethal damage repair, or PLDR, and is an increase in the ultimate survival of cells incubated

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Fig. 9. Radiation-induced cytotoxicity in drug-treated HeLa cells. Cells were exposed to 0.79 mM novobiocin ([J), 1.58 mM novobiocin (I), 5 mM caffeine (e) or left unexposed (©) for 60 min before, and for 4 h after, exposure to increasing radiation doses (as indicated). Cells were washed free of drug and single cell survival assayed as described in the Methods section. Insert: Cells were drug-treated for 60 min before, and between, a split radiation dose of 8 (e) or 10 ( o , E]) Gy which was divided by the time periods indicated. The symbols are the same as in A.

immediately after irradiation under conditions (e.g., at 2 5 ° C or in physiological saline) which inhibit cell cycle progression. When HeLa cells were left at 2 5 ° C for up to 5 h after irradiation there was no survival increase. Thus no P L D R was expressed in these cells. To determine the effect(s) of exposure to novobiocin or caffeine on PLDR we made use of a hamster cell line. When

53 Chinese hamster ovary (CHO) cells were exposed to 8 G y at 25 ° C (a surviving fraction of 0.01) and left at 2 5 ° C for increasing time periods before being returned to 37 ° C, an increase in survival was observed with a maximum survival increase of 1.6 which developed with a half-time of about 60 min (results not shown). In a manner similar to that observed for H e L a cell SLDR, exposure to 0.79 mM novobiocin had no detectable effect on the development of P L D R in C H O cells, while exposure to 5 m M caffeine completely inhibited its development. Discussion The purpose of the present work was to study the role, if any, of the topoisomerase enzymes in the response of mammalian cells to ionizing radiation. Two types of topoisomerase enzymes are routinely detected in mammalian cells: those which produce topological changes in D N A by the production of a D N A single-strand nick (type-I enzymes) and those which do so by the production of a D N A double-strand cut (type-II enzymes). These characteristically different activities enable a distinction between the two enzyme types by their in vitro action on supercoiled plasmid D N A (Fig. 1, insert). An estimation of the capacity of radiation to alter the level of these two enzyme activities in sonicates of H e L a cells indicated that the relaxation activity (predominantly topo I) and catenation activity (topo II) were unaffected even after exposure to extremely large radiation doses. The capacity of topo II to form a cleavable complex with D N A in the presence of the drug mAMSA was unaffected by exposure to 10 G y of X-radiation. In addition, the total topo II activity measurable in cells remained constant for up to 90 min after X-irradiation. We conclude that at biologically relevant doses, i.e., doses equal to, or less than, 10 G y where some cell survival can be detected, the total cellular activity of these two enzymes remains virtually unchanged for up to 90 rain after radiation exposure. Since both enzyme activities remained unaffected by radiation exposure we could ask their role in the cell's response to ionizing radiation only through the device of drug-induced enzyme inhibition. Only in the case of the type-II topo

enzyme are there drugs such as novobiocin or caffeine available which produce no D N A lesions and effectively inhibit this enzyme at nontoxic concentrations. In H e L a cells, using a plasmid catenation assay (Fig. 3), we found that topo II activity was 50% inhibited by exposure to 0.79 m M novobiocin while exposure to 1.58 m M of the drug was required before the H e L a cell topo II enzyme activity was predominantly (90%) inhibited. In preliminary studies with C H O cells we find approximately the same degree of inbibition of cell metabolism ( D N A and R N A synthesis) per drug exposure level. While in vitro assays of topo II drug sensitivity may not extrapolate directly to the effect of these drugs on endogenous, cellular topo II activities, the results indicate that greater concentrations of novobiocin than previously have been used are required to inhibit a significant fraction of topo II in exponentially growing m a m malian cells. Another drug which inhibited topo II activity at concentrations which produced neither D N A damage nor cytotoxicity was caffeine. Caffeine inhibited plasmid catenation by about 40% at 5 mM, and both k D N A decatenation and cleavable complex formation by 50% at 15 mM, but had no apparent effect on relaxation of supercoiled plasmid DNA. So this drug does not appear to proclude the interaction of topoisomerase enzymes with D N A per se. Rather it appears to inhibit topoisomerase II at a stage prior to the enzyme's ATP-dependent, D N A strand passage activity. A comparison of these two drugs was especially interesting since they had similar effects on D N A repair, but dramatically different effects on radiation cytotoxicity. Both drugs inhibited the reconstitution in irradiated cells of D N A supercoiling (nucleoid sedimentation) to the same extent that they inhibited topo II activity (cleavable complex formation). Neither drug, however, affected the initial rate of D N A single- or double-strand break rejoining. These results indicate that while topo II activity may be integral to the reconstruction of D N A supercoiling subsequent to cell irradiation, topo II activity is not an absolute requirement for the removal of the majority of D N A strand breaks. Since in the presence of both drugs D N A strand break rejoining proceeded at a normal rate, while nucleoid reconstitution was inhibited, the recon-

54 stitution of D N A supercoiling in i r r a d i a t e d cells m u s t not be an integral r e q u i r e m e n t for D N A s t r a n d break removal. It seems highly likely, however, that the converse m u s t be true; that D N A s t r a n d b r e a k r e m o v a l is an a b s o l u t e r e q u i r e m e n t for D N A supercoiling to be p r o p e r l y r e c o n s t i t u t e d in i r r a d i a t e d cells. R a d i a t i o n - i n d u c e d c y t o t o x i c i t y was not detect a b l y affected b y c o n t i n u o u s e x p o s u r e to 0.79 m M novobiocin. Caffeine (5 m M ) however signific a n t l y increased r a d i a t i o n - i n d u c e d cytotoxicity, p r e s u m a b l y by inhibiting the s h o r t - t e r m recovery ( S L D R ) of cells from r a d i a t i o n d a m a g e (Fig. 9). The p r o d u c t i o n b y ionizing r a d i a t i o n of lesions in the cell nucleus ( W a r t e r s et al., 1977), most p r o b a b l y D N A d o u b l e - s t r a n d b r e a k s (Ritter et al., 1977; R o o t s et al., 1979; K e m p et al., 1984; R a d ford, 1985), is c u r r e n t l y thought to be the cellular lesion p r i m a r i l y r e s p o n s i b l e for r a d i a t i o n - i n d u c e d cytotoxicity. Since e x p o s u r e to neither drug affected the initial rate of r e m o v a l of D N A lesions, yet had d r a m a t i c a l l y different consequences for s h o r t - t e r m cellular recovery from r a d i a t i o n exposure, the results i n d i c a t e a negative correlation between the frequency of D N A lesions d e t e c t a b l e b y these c o n v e n t i o n a l D N A d a m a g e assays a n d the s h o r t - t e r m c a p a c i t y of cells to recover from r a d i a t i o n injury. This conclusion is also i n d i c a t e d by the finding that while D N A d o u b l e - s t r a n d b r e a k rejoining was significantly i n h i b i t e d in cells e x p o s e d to 1.58 m M novobiocin, cellular recovery from sublethal d a m a g e over the first 4 h after i r r a d i a t i o n was as r a p i d a n d extensive as in control cells (results n o t shown). The results d o not argue against an integral role of r a d i a t i o n - i n d u c e d D N A lesions in the l o n g - t e r m survival of cells after r a d i a t i o n exposure, reflected p r e d o m i n a n t l y by the slope of the survival curve. In conclusion, the total cellular activity of neither t o p o i s o m e r a s e I nor II was significantly affected b y a w i d e - r a n g e r a d i a t i o n exposure, n o r d i d t o p o II activity change d u r i n g the first 90 min of cellular recovery from r a d i a t i o n exposure. Inh i b i t i o n b y e x p o s u r e to the d r u g n o v o b i o c i n of the ATP-dependent f u n c t i o n s u n i q u e to t o p o isomerase II d i d n o t alter the cell's c a p a c i t y to r e m o v e D N A lesions or recover from r a d i a t i o n injury. Therefore, these results suggest that the A T P - d e p e n d e n t t o p o i s o m e r a s e II activities such as

D N A c a t e n a t i o n a n d d e c a t e n a t i o n a n d reconstitution of D N A supercoiling are not integral to the cell's early response to ionizing r a d i a t i o n . Inhibition of t o p o i s o m e r a s e I1 at a stage p r i o r to its A T P - d e p e n d e n t functions by e x p o s u r e to caffeine h a d no affect on D N A d a m a g e r e m o v a l but b l o c k e d the cell's c a p a c i t y to recover from radiation injury. These c o n t r a s t i n g o b s e r v a t i o n s suggest that if t o p o i s o m e r a s e II is involved at all in the cell's response to ionizing r a d i a t i o n , this requirem e n t is m e d i a t e d through a n o n - A T P - d e p e n d e n t enzyme activity.

Acknowledgements The a u t h o r s w o u l d like to t h a n k Ms. M a r y H o g a n for t y p i n g the m a n u s c r i p t a n d Drs. Lyle A. Dethlefsen a n d C h r i s t o p h e r M. L e h m a n for critical review of the m a n u s c r i p t . This w o r k was s u p p o r t e d b y N 1 H g r a n t C A 25957.

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Roots, R., T.C. Yang, L. Craise, E.A. Blakely and C.A. Tobias (1979) Impaired repair capacity of DNA breaks induced in mammalian cell DNA by accelerated heavy ions, Radiat. Res., 78, 38-49. Rowley, R., and L. Kort (1988) Effect of caffeine on the repair of radiation-induced DNA damage detectable by neutral filter elution, Radiat. Res., in press. Sandhu, L.C., R.L. Warters and L.A. Dethlefsen (1985) Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA, Cytometry, 6, 191-194. Simpson, A.M., and L. Simpson (1974) Isolation and characterization of kinetoplast DNA networks and minicircles from Crithidia fasciculata, J. Protozool., 21,774 781. Warters, R.L., and K.J. Henle (1982) DNA degradation in Chinese hamster ovary cells after exposure to hyperthermia, Cancer Res., 42, 4427-4432. Warters, R.L., K.G. Hofer, C.R. Harris and J.M. Smith (1977) Radionuclide toxicity in cultured mammalian cells: Elucidation of the primary site of radiation damage, Curr. Topics Radiat. Res., Quart., 12, 389-407. Warters, R.L., L.M. Brizgys and J. Axtell-Bartlett (1985a) DNA damage production in CHO cells at elevated temperatures, J. Cell. Physiol., 124, 481-486. Warters, R.L, B.W. Lyons, D.N. Ridinger and L.A. Dethlefsen (1985b) DNA damage repair in quiescent murine mammary carcinoma cells in culture, Biochim. Biophys. Acta, 824, 357-364. Warters, R.L., B.W. Lyons, S.M. Chiu and N.L. Oleinick (1987) Induction of DNA strand breaks in transcriptionally active DNA sequences of mouse cells by low doses of ionizing radiation, Mutation Res., 180, 21-29. Weibezahn, K.F., and T.M. Coquerelle (1986) Relationship between double strand break rejoining and G 2 block formation in V79 cells, Radiat. Environ. Biophys., 25, 13-21. Zwelling, L.A., S. Michaels, L.C. Erickson, R.S. Ungerleider, M. Nichols and K.W. Kohn (1981) Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4'-(9acridinylamino)-methanesulfon-m-anisidide and adriamycin, Biochemistry, 20, 6553 6563.