167
Mutation Research, DNA Repair, 254 (1991) 167-174 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100058X
MUTDNA 06421
Topoisomerase II activity in a D N A double-strand break repair deficient Chinese hamster ovary cell line Raymond L. Warters a, Bradley W. Lyons a, T. Mua Li
a
and David J. Chen b
a Department of Radiology, University of Utah Health Sciences Center, Salt Lake City, UT84132 and b Genetics Group, Los Alamos National Laboratory, Los Alamos, N M 87545 (U.S.A.)
(Received 5 February 1990) (Revision received 24 July 1990) (Accepted 24 July 1990)
Keywords: DNA double-strand break repair; Topoisomerase II; Chinese hamster ovary cell line
Summary Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiationinduced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.
Mammalian cells contain enzymes termed topoisomerases which are likely required to deal with the changes in DNA conformation and topology which occur during the replication and transcription of nuclear DNA. Two types of topoisomerase enzymes have been isolated from mammalian cells, distinguished by their ability to introduce transient, protein-bridged DNA breaks on one (type I) or both (type II) DNA strands (reviewed by Gellert, 1981, and Wang, 1985).
Correspondence: Raymond L. Warters, Ph.D., Department of Radiology, University of Utah Health Sciences Center, Salt Lake City, UT 84132 (U.S.A.).
While either topoisomerase can relax supercoiled DNA, only the type II enzyme can catenate or decatenate two intact DNA circles by virtue of this enzyme's DNA strand passing activity associated with ATP hydrolysis (Miller et al., 1981). The type II DNA topoisomerase enzyme has been found to be the nuclear target of a number of anticancer drugs (Liu, 1989). These drugs include DNA intercalative drugs such as adriamycin, e!lipticine and rnitoxanthrone (Zwelling et al., 1981; Pommier et al., 1983; Tewey et al., 1984) as well as nonintercalative glycosidic derivatives of the podophyllotoxins such as VP-16 and VM-26 (Wozniak and Ross, 1983; Long et al., 1984). These drugs interfere with the breakage-rejoining
168
activity of the enzyme and simultaneously trap the enzyme in a DNA-associated form termed a cleavable complex in which the enzyme is covalently linked to the 5'-phosphoryl end of each broken D N A strand via a phosphotyrosyl linkage (Rowe et al., 1986). The basis for the cytotoxicity of these drugs as yet has not been demonstrated. One likely speculation is that some aspect of the protein-associated, single or double DNA-strand breaks induced by these drugs results in cytotoxicity. While exposure to these drugs does result in chromosome-aberration formation and sisterchromatid exchanges (Pommier et al., 1985; Dillehay et al., 1987) a direct correlation between the DNA-strand breaks and cytotoxicity is not routinely obtained (Zwelling et al., 1981; Zwelling et al., 1982; Rowe et al., 1983; Covey et al., 1988). One approach for assessing the role of DNAstrand break induction in the cytotoxicity of these topoisomerase poisons is by studying the interaction of these drugs with a DNA-damage repair deficient cell line such as the rodent xrs-6 cell line. This cell line was originally isolated from Chinese hamster ovary kl cells and was found to be extraordinarily sensitive to ionizing radiation (Jeggo and Kemp, 1983). The xrs-6 cell line subsequently was found to be proficient in the rejoining of single, but deficient in the rejoining of double, DNA-strand breaks (Kemp et al., 1984; Weibezahn et al., 1985). This xrs-6 cell line was chosen in these studies to examine the potential role of D N A double-strand break formation in the cytotoxicity of the topoisomerase II poison m-AMSA. Materials and methods
Cell culture and labeling Chinese hamster ovary (CHO) K1 cells, and the radiation sensitive CHO xrs-6 cells, were grown in monolayer culture in McCoy's medium 5A supplemented with 5% fetal bovine and 5% calf serum. Monolayer cultures were uniformly labeled in their D N A by exposure for 18 h to whole medium containing [Me-14C]thymidine at 0.07 ~Ci/ml. Cells were exposed to X-irradiation with a Philips model RT 250 X-ray unit at 250 kVp, 15 mA, with 3-mm aluminum filtration at a dose rate of 5.0 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 DNA single-strand break induction was measured by the alkaline (pH 12.2) filter elution assay, and D N A double-strand break induction by the neutral (pH 7.2) filter-elution assay, as previously described (Warters et al., 1989; Flick et al., 1989). The size of ssDNA was estimated by sedimentation of whole-cell D N A through alkaline (pH 12.2) sucrose gradients, as described previously (Warters et al., 1989). Topoisomerase H extraction Topoisomerase II was extracted from cells as previously described (Miller et al., 1981). Approximately 108 hamster cells were resuspended in a permeabilization buffer (150 mM NaC1, 1 mM K H 2 P O 4, pH 6.4, 5 mM MgC12, 10 mM EDTA and 0.05 mM dithiothreitol) and immediately pelleted. The cells were resuspended in 0.5 vol. of this buffer and 4.5 vol. of this buffer containing 0.3% Triton X-100. The cells were left in this buffer for 10 min at 4°C at which time 100% of the cells were permeable to the dye trypan blue. The resulting nuclei were pelleted and resuspended in 1 ml of the permeabilization buffer. The supernatant over a 1.5 M NaC1, 4% PEG extraction of these nuclei was dialyzed for 24 h into a buffer containing 10% glycerol, 40 mM Tris-HC1, pH 7.5, 10 m M fl-mercaptoethanol, 1 mM PMSF. Topoisomerase H assays The decatenation activity in hamster cell nuclear extracts was measured using kinetoplast D N A as a substrate. Kinetoplast D N A networks were isolated as previously described (Miller et al., 1981) from the trypanosome Crithidia fasciculata cultured in Brain Heart Infusion medium (Difco) supplemented with 1 0 / ~ g / m l hemin (Sigma). The kinetoplast D N A was prepared from exponential phase cultures and purified on cesium chlorideethidium bromide density gradients as described by Simpson and Simpson (1974). Kinetoplast D N A was labeled to 2 x 10 4 d p m / # g by culture of Crithidia fasciculata in 2 btCi/ml [3H]thymidine for 6 h. Topoisomerase II activity was considered an ATP-dependent, novobiocin-sensitive, de-
169 catenation activity. A standard decatenation reaction (28 /~1) contained 1-5 #1 of the enriched topoisomerase II, 21-25/tl of a reaction buffer (50 mM Tris-HCl, pH 8.0, 120 mM KC1, 10 mM MgC12, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 /~l/ml BSA and 0.5 mM ATP) and 2 /~1 of kinetoplast DNA (0.5-1.0 /tg DNA total). The reaction mixture was placed at 37 °C for 60 rain. The topological states of the kinetoplast DNA were assessed by horizontal electrophoresis in a 1% agarose gel at 1.5 V / c m for 18 h, as previously described (Wafters et al., 1989). In some cases the capacity of nuclear extracts to catenate supercoiled, plasmid pBR322 was determined, as described previously (Warters et al., 1989). Alternately, the decatenation of 3H-labeled, kinetoplast DNA by nuclear extracts was determined by a filter-collection assay described elsewhere (Holden and Low, 1985).
ioo
rn -AMSA (p.M) I 2 I I
3 I
4 I
io-I. ...1
n-. r..o ._J ,,~
io-2_
z o I.rr la_
io-3_
Results
Exposure of K1 or xrs-6 Chinese hamster ovary cells to increasing concentrations of the DNA-intercalating drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) for 60 min at 37 o C resulted in cytotoxicity in both cell lines (Fig. 1). At lower drug concentrations the xrs-6 cells were approximately 3 times more sensitive to drug-induced cytotoxicity than the K1 cells. The reciprocal slope for drug-induced cytotoxicity of the xrs-6, or K1 cells, was 0.2 and 0.6/~M m-AMSA, respectively. At higher concentrations a drug-resistant tail appeared for xrs-6 drug toxicity indicating that a small portion (approximately 5%) of the xrs-6 cells may exhibit a sensitivity to m-AMSA equivalent to that expressed by the wild-type, K1 cells. The DNA-intercalating drug m-AMSA interacts with topoisomerase II by inhibiting its DNAstrand passage activity. This interaction results in an inhibition of the topoisomerase II decatenation activity in vitro (Warters et al., 1989) and in the production of protein-associated, DNA-strand breaks in the cell's genome (Zwelling et al., 1981; Nelson et al., 1984, Minford et al., 1986). Thus the activity of topoisomerase II in the intact cell nucleus can be estimated by determining the extent of drug-induced breakage of deproteinized
io-4_ Fig. 1. Drug-induced cytotoxicity in K1 and xrs-6 cells. Exponential monolayer cultures of K1 (o) or xrs-6 (D) hamster cells were exposed to increasing concentrations of the drug m-AMSA at 37 ° C for 60 min. The cells were washed free of the drug by two consecutive 60-rain exposures to drug-free medium at 37 o C. Additional drug-free medium was added and cell survival assayed as described in the Methods section, The average + one standard deviation is plotted in this, ~nd subsequent, figures.
DNA from m-AMSA exposed cells. K1 and xrs-6 cells were exposed to increasing concentrations of the drug m-AMSA, and their DNA analyzed for the presence of DNA single-strand breakage at alkaline pH (Fig. 2, Panel A) or DNA doublestrand breakage at neutral pH (Fig. 2, Panel B). At all drug concentrations somewhat fewer (2530%) DNA single- or double-strand breaks were detected in xrs-6 than in K1 hamster cells. When the two hamster cell lines were exposed to increasing concentrations of m-AMSA from 0.1 to 10.0 mM and their nuclear DNA analyzed by sedimentation through alkaline sucrose gradients, DNA size progressively decreased (i.e., the TCA-insoluble DNA radioactivity sedimented progressively
170
less far into the gradients) (results not shown). No further reduction in ssDNA length was detected with exposure to m-AMSA concentrations greater than 10 /~M. In both cell lines the majority of D N A had a length estimated between 1 and 3 x 105 nucleotides after exposure to 10 ffM m-AMSA. Total topoisomerase II activity in K1 or xrs-6 nuclei was estimated by the capacity of nuclear extracts to decatenate kinetoplast D N A networks, as estimated by electrophoresis through 1% agar gels (Fig. 3). When the cytoplasmic fraction over pelleted nuclei was dialyzed overnight and topoisomerase II activity estimated by the decatenation reaction, greater than 98% of the total detectable cellular enzyme activity remained in the residual nuclear protein fraction. When the residual nuclear protein which was either extracted during exposure to 1.5 M NaC1 or remained in the insoluble, and subsequently sonicated, nuclear protein matrix was dialyzed overnight and assayed by a decatenation assay, greater than 95% of the detectable topoisomerase II routinely was found in the salt-extracted nuclear protein. Thus we expect that in excess of 90% of cellular topoisomerase II activity was recovered in the 1.5 M NaC1 extraction of
1.2 A. pH 12.2
O
Ki
XRS-6
Fig. 3. Topoisomerase II activity in K1 or xrs-6 cells. The protein from l0 s KI or xrs-6 nuclei, prepared as described in the Methods section, was isolated by 1.5 M NaCI, 4% PEG 4000 extraction. Topoisomerase II activity in these samples was determined by their ability to decatenate, during a 60-min incubation at 3 7 ° C , 1 /tg of kinetoplast D N A isolated from Crithidia fasciculata. Kinetoplast D N A networks (N) will not enter a 1% agar gel. Increasing equivalents (as indicated) of K1 (top gel) or xrs-6 (bottom gel) nuclear extract decatenate the k D N A network into its constituent m o n o m e r circles (M) which do enter the gel.
-lB. pH 7.2
1.0 0.8 0.6 0.4" 0.20.0
0.0
0.2
0.4 0.6 0.8 0.0 2.0 40 rn-AMSA CONCENTRATION (I~M)
60
80
Fig. 2. Drug-induced D N A strand breakage in K1 and xrs-6 cells. Exponentially growing K1 (O) or xrs-6 (m) cells were exposed to increasing concentrations (as indicated) of the DNA-intercalating drug m - A M S A at 3 7 ° C for 60 min. The cells were collected by trypsinization and their D N A analyzed by filter elution, as described previously (Flick et al., 1989; Warters et al., 1989), at pH 12.2 for the presence of D N A single-strand breaks (Panel A) or at p H 7.2 for the presence of D N A double-strand breaks (Panel B). A D N A strand-scission factor (SSF), calculated as described by Flick et al. (1989), is plotted versus the drug exposure concentration.
nuclei and assayed in these experiments. Between 0.5 and 1 x 10 4 hamster cell nuclear equivalents of topoisomerase II was required to fully decatenate 1 /xg of k D N A within 60 min at 37°C. While the total decatenation activity extracted from either K1 or xrs-6 nuclei varied by as much as 20% over 5 Expts., the average, total topoisomerase II activity per K1 or xrs-6 nucleus appeared to be identical (Fig. 3). Similar results were obtained when the capacity of these K1 or xrs-6 nuclear extracts to catenate plasmid pBR322 D N A was determined by agar gel electrophoresis. In addition the capacity of K1 or xrs-6 nuclear ex-
171 or.
u.i
.0-
h Z 0
0.8-
i.-
5
0.6-
0
=
Mutation Research, DNA Repair, 254 (1991) 167-174 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100058X
MUTDNA 06421
Topoisomerase II activity in a D N A double-strand break repair deficient Chinese hamster ovary cell line Raymond L. Warters a, Bradley W. Lyons a, T. Mua Li
a
and David J. Chen b
a Department of Radiology, University of Utah Health Sciences Center, Salt Lake City, UT84132 and b Genetics Group, Los Alamos National Laboratory, Los Alamos, N M 87545 (U.S.A.)
(Received 5 February 1990) (Revision received 24 July 1990) (Accepted 24 July 1990)
Keywords: DNA double-strand break repair; Topoisomerase II; Chinese hamster ovary cell line
Summary Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiationinduced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.
Mammalian cells contain enzymes termed topoisomerases which are likely required to deal with the changes in DNA conformation and topology which occur during the replication and transcription of nuclear DNA. Two types of topoisomerase enzymes have been isolated from mammalian cells, distinguished by their ability to introduce transient, protein-bridged DNA breaks on one (type I) or both (type II) DNA strands (reviewed by Gellert, 1981, and Wang, 1985).
Correspondence: Raymond L. Warters, Ph.D., Department of Radiology, University of Utah Health Sciences Center, Salt Lake City, UT 84132 (U.S.A.).
While either topoisomerase can relax supercoiled DNA, only the type II enzyme can catenate or decatenate two intact DNA circles by virtue of this enzyme's DNA strand passing activity associated with ATP hydrolysis (Miller et al., 1981). The type II DNA topoisomerase enzyme has been found to be the nuclear target of a number of anticancer drugs (Liu, 1989). These drugs include DNA intercalative drugs such as adriamycin, e!lipticine and rnitoxanthrone (Zwelling et al., 1981; Pommier et al., 1983; Tewey et al., 1984) as well as nonintercalative glycosidic derivatives of the podophyllotoxins such as VP-16 and VM-26 (Wozniak and Ross, 1983; Long et al., 1984). These drugs interfere with the breakage-rejoining
168
activity of the enzyme and simultaneously trap the enzyme in a DNA-associated form termed a cleavable complex in which the enzyme is covalently linked to the 5'-phosphoryl end of each broken D N A strand via a phosphotyrosyl linkage (Rowe et al., 1986). The basis for the cytotoxicity of these drugs as yet has not been demonstrated. One likely speculation is that some aspect of the protein-associated, single or double DNA-strand breaks induced by these drugs results in cytotoxicity. While exposure to these drugs does result in chromosome-aberration formation and sisterchromatid exchanges (Pommier et al., 1985; Dillehay et al., 1987) a direct correlation between the DNA-strand breaks and cytotoxicity is not routinely obtained (Zwelling et al., 1981; Zwelling et al., 1982; Rowe et al., 1983; Covey et al., 1988). One approach for assessing the role of DNAstrand break induction in the cytotoxicity of these topoisomerase poisons is by studying the interaction of these drugs with a DNA-damage repair deficient cell line such as the rodent xrs-6 cell line. This cell line was originally isolated from Chinese hamster ovary kl cells and was found to be extraordinarily sensitive to ionizing radiation (Jeggo and Kemp, 1983). The xrs-6 cell line subsequently was found to be proficient in the rejoining of single, but deficient in the rejoining of double, DNA-strand breaks (Kemp et al., 1984; Weibezahn et al., 1985). This xrs-6 cell line was chosen in these studies to examine the potential role of D N A double-strand break formation in the cytotoxicity of the topoisomerase II poison m-AMSA. Materials and methods
Cell culture and labeling Chinese hamster ovary (CHO) K1 cells, and the radiation sensitive CHO xrs-6 cells, were grown in monolayer culture in McCoy's medium 5A supplemented with 5% fetal bovine and 5% calf serum. Monolayer cultures were uniformly labeled in their D N A by exposure for 18 h to whole medium containing [Me-14C]thymidine at 0.07 ~Ci/ml. Cells were exposed to X-irradiation with a Philips model RT 250 X-ray unit at 250 kVp, 15 mA, with 3-mm aluminum filtration at a dose rate of 5.0 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 DNA single-strand break induction was measured by the alkaline (pH 12.2) filter elution assay, and D N A double-strand break induction by the neutral (pH 7.2) filter-elution assay, as previously described (Warters et al., 1989; Flick et al., 1989). The size of ssDNA was estimated by sedimentation of whole-cell D N A through alkaline (pH 12.2) sucrose gradients, as described previously (Warters et al., 1989). Topoisomerase H extraction Topoisomerase II was extracted from cells as previously described (Miller et al., 1981). Approximately 108 hamster cells were resuspended in a permeabilization buffer (150 mM NaC1, 1 mM K H 2 P O 4, pH 6.4, 5 mM MgC12, 10 mM EDTA and 0.05 mM dithiothreitol) and immediately pelleted. The cells were resuspended in 0.5 vol. of this buffer and 4.5 vol. of this buffer containing 0.3% Triton X-100. The cells were left in this buffer for 10 min at 4°C at which time 100% of the cells were permeable to the dye trypan blue. The resulting nuclei were pelleted and resuspended in 1 ml of the permeabilization buffer. The supernatant over a 1.5 M NaC1, 4% PEG extraction of these nuclei was dialyzed for 24 h into a buffer containing 10% glycerol, 40 mM Tris-HC1, pH 7.5, 10 m M fl-mercaptoethanol, 1 mM PMSF. Topoisomerase H assays The decatenation activity in hamster cell nuclear extracts was measured using kinetoplast D N A as a substrate. Kinetoplast D N A networks were isolated as previously described (Miller et al., 1981) from the trypanosome Crithidia fasciculata cultured in Brain Heart Infusion medium (Difco) supplemented with 1 0 / ~ g / m l hemin (Sigma). The kinetoplast D N A was prepared from exponential phase cultures and purified on cesium chlorideethidium bromide density gradients as described by Simpson and Simpson (1974). Kinetoplast D N A was labeled to 2 x 10 4 d p m / # g by culture of Crithidia fasciculata in 2 btCi/ml [3H]thymidine for 6 h. Topoisomerase II activity was considered an ATP-dependent, novobiocin-sensitive, de-
169 catenation activity. A standard decatenation reaction (28 /~1) contained 1-5 #1 of the enriched topoisomerase II, 21-25/tl of a reaction buffer (50 mM Tris-HCl, pH 8.0, 120 mM KC1, 10 mM MgC12, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 /~l/ml BSA and 0.5 mM ATP) and 2 /~1 of kinetoplast DNA (0.5-1.0 /tg DNA total). The reaction mixture was placed at 37 °C for 60 rain. The topological states of the kinetoplast DNA were assessed by horizontal electrophoresis in a 1% agarose gel at 1.5 V / c m for 18 h, as previously described (Wafters et al., 1989). In some cases the capacity of nuclear extracts to catenate supercoiled, plasmid pBR322 was determined, as described previously (Warters et al., 1989). Alternately, the decatenation of 3H-labeled, kinetoplast DNA by nuclear extracts was determined by a filter-collection assay described elsewhere (Holden and Low, 1985).
ioo
rn -AMSA (p.M) I 2 I I
3 I
4 I
io-I. ...1
n-. r..o ._J ,,~
io-2_
z o I.rr la_
io-3_
Results
Exposure of K1 or xrs-6 Chinese hamster ovary cells to increasing concentrations of the DNA-intercalating drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) for 60 min at 37 o C resulted in cytotoxicity in both cell lines (Fig. 1). At lower drug concentrations the xrs-6 cells were approximately 3 times more sensitive to drug-induced cytotoxicity than the K1 cells. The reciprocal slope for drug-induced cytotoxicity of the xrs-6, or K1 cells, was 0.2 and 0.6/~M m-AMSA, respectively. At higher concentrations a drug-resistant tail appeared for xrs-6 drug toxicity indicating that a small portion (approximately 5%) of the xrs-6 cells may exhibit a sensitivity to m-AMSA equivalent to that expressed by the wild-type, K1 cells. The DNA-intercalating drug m-AMSA interacts with topoisomerase II by inhibiting its DNAstrand passage activity. This interaction results in an inhibition of the topoisomerase II decatenation activity in vitro (Warters et al., 1989) and in the production of protein-associated, DNA-strand breaks in the cell's genome (Zwelling et al., 1981; Nelson et al., 1984, Minford et al., 1986). Thus the activity of topoisomerase II in the intact cell nucleus can be estimated by determining the extent of drug-induced breakage of deproteinized
io-4_ Fig. 1. Drug-induced cytotoxicity in K1 and xrs-6 cells. Exponential monolayer cultures of K1 (o) or xrs-6 (D) hamster cells were exposed to increasing concentrations of the drug m-AMSA at 37 ° C for 60 min. The cells were washed free of the drug by two consecutive 60-rain exposures to drug-free medium at 37 o C. Additional drug-free medium was added and cell survival assayed as described in the Methods section, The average + one standard deviation is plotted in this, ~nd subsequent, figures.
DNA from m-AMSA exposed cells. K1 and xrs-6 cells were exposed to increasing concentrations of the drug m-AMSA, and their DNA analyzed for the presence of DNA single-strand breakage at alkaline pH (Fig. 2, Panel A) or DNA doublestrand breakage at neutral pH (Fig. 2, Panel B). At all drug concentrations somewhat fewer (2530%) DNA single- or double-strand breaks were detected in xrs-6 than in K1 hamster cells. When the two hamster cell lines were exposed to increasing concentrations of m-AMSA from 0.1 to 10.0 mM and their nuclear DNA analyzed by sedimentation through alkaline sucrose gradients, DNA size progressively decreased (i.e., the TCA-insoluble DNA radioactivity sedimented progressively
170
less far into the gradients) (results not shown). No further reduction in ssDNA length was detected with exposure to m-AMSA concentrations greater than 10 /~M. In both cell lines the majority of D N A had a length estimated between 1 and 3 x 105 nucleotides after exposure to 10 ffM m-AMSA. Total topoisomerase II activity in K1 or xrs-6 nuclei was estimated by the capacity of nuclear extracts to decatenate kinetoplast D N A networks, as estimated by electrophoresis through 1% agar gels (Fig. 3). When the cytoplasmic fraction over pelleted nuclei was dialyzed overnight and topoisomerase II activity estimated by the decatenation reaction, greater than 98% of the total detectable cellular enzyme activity remained in the residual nuclear protein fraction. When the residual nuclear protein which was either extracted during exposure to 1.5 M NaC1 or remained in the insoluble, and subsequently sonicated, nuclear protein matrix was dialyzed overnight and assayed by a decatenation assay, greater than 95% of the detectable topoisomerase II routinely was found in the salt-extracted nuclear protein. Thus we expect that in excess of 90% of cellular topoisomerase II activity was recovered in the 1.5 M NaC1 extraction of
1.2 A. pH 12.2
O
Ki
XRS-6
Fig. 3. Topoisomerase II activity in K1 or xrs-6 cells. The protein from l0 s KI or xrs-6 nuclei, prepared as described in the Methods section, was isolated by 1.5 M NaCI, 4% PEG 4000 extraction. Topoisomerase II activity in these samples was determined by their ability to decatenate, during a 60-min incubation at 3 7 ° C , 1 /tg of kinetoplast D N A isolated from Crithidia fasciculata. Kinetoplast D N A networks (N) will not enter a 1% agar gel. Increasing equivalents (as indicated) of K1 (top gel) or xrs-6 (bottom gel) nuclear extract decatenate the k D N A network into its constituent m o n o m e r circles (M) which do enter the gel.
-lB. pH 7.2
1.0 0.8 0.6 0.4" 0.20.0
0.0
0.2
0.4 0.6 0.8 0.0 2.0 40 rn-AMSA CONCENTRATION (I~M)
60
80
Fig. 2. Drug-induced D N A strand breakage in K1 and xrs-6 cells. Exponentially growing K1 (O) or xrs-6 (m) cells were exposed to increasing concentrations (as indicated) of the DNA-intercalating drug m - A M S A at 3 7 ° C for 60 min. The cells were collected by trypsinization and their D N A analyzed by filter elution, as described previously (Flick et al., 1989; Warters et al., 1989), at pH 12.2 for the presence of D N A single-strand breaks (Panel A) or at p H 7.2 for the presence of D N A double-strand breaks (Panel B). A D N A strand-scission factor (SSF), calculated as described by Flick et al. (1989), is plotted versus the drug exposure concentration.
nuclei and assayed in these experiments. Between 0.5 and 1 x 10 4 hamster cell nuclear equivalents of topoisomerase II was required to fully decatenate 1 /xg of k D N A within 60 min at 37°C. While the total decatenation activity extracted from either K1 or xrs-6 nuclei varied by as much as 20% over 5 Expts., the average, total topoisomerase II activity per K1 or xrs-6 nucleus appeared to be identical (Fig. 3). Similar results were obtained when the capacity of these K1 or xrs-6 nuclear extracts to catenate plasmid pBR322 D N A was determined by agar gel electrophoresis. In addition the capacity of K1 or xrs-6 nuclear ex-
171 or.
u.i
.0-
h Z 0
0.8-
i.-
5
0.6-
0
=
