- Email: [email protected]
Sensitivity to inhibitors of type II topoisomerases from mouse L5178Y lymphoma strains that are resistant or sensitive to X-radiation
Mutation Research, 285 (1993) 175-179 © 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00
175
MUT 05193
Sensitivity to inhibitors of type II topoisomerases from mouse L5178Y lymphoma strains that are resistant or sensitive to X-radiation Krzysztof Staron a, Barbara Kowalska-Loth a, Irena Szumiel b and Iwona Buraczewska b a Institute of Biochemistry, Warsaw University, Warsaw, Poland and ~ Institute of Nuclear Chemistry and Technology, Warsaw, Poland (Received 26 March 1992) (Revision received 4 August 1992) (Accepted 6 August 1992)
Keywords: Topoisomerase II inhibitors; L5178Y mouse lymphoma cells
Summary Sensitivity to topoisomerase II inhibitors was tested at the cellular and enzyme level for two strains of mouse L5178Y lymphoma cells: resistant (LY-R) and sensitive (LY-S) to X-radiation. Differences in the susceptibility to inhibitors between LY-R and LY-S cells depended on the inhibitor used and were observed for adriamycin and VP-16, but not for mitoxantrone. On the other hand, isolated enzymes displayed the same sensitivity to all inhibitors tested regardless of the cell line. These results exclude the presence of altered topoisomerase II in LY-S cells as a possible reason for the collateral sensitivity of LY-S cells to X-radiation and topoisomerase II inhibitors.
Two strains of mouse lymphoma L5178Y cells, which differ in their sensitivity to X-radiation (Alexander and Mikulski, t961), have been found to be differently sensitive to topoisomerase II inhibitors: m-AMSA, VP-16 and ellipticine (Evans et al., 1989). Strain LY-S, which is sensitive to X-radiation compared to strain LY-R, is also more sensitive to the above inhibitors of topoisomerase II. The concomitance of radiation sensitivity and susceptibility to topoisomerase II poisons has been suggested to result either (1) from altered topoisomerase II in strain LY-S or (2) from the DNA double-strain break repair deficiency in that strain (Evans et al., 1989). The aim
Materials Drugs used in this work were commercial pharmaceutical preparations: adriamycin (Adriblastina; Farmitalia, Carlo Erba, Milan, Italy), mitoxantrone (Novatron; Cyanamid GmbH, Wolfratshausen, Germany) and VP-16 (Vepesid; Bristol-Myers GmbH, Bergisch-Gladbach, Germany).
Correspondence: Krzysztof Staron, Institute of Biochemistry, Warsaw University, AI. Zwirki i Wigury 93, 02-089 Warsaw, Poland.
Cell culture LY-R and -S cells were cultured in suspension in Fischer's medium supplemented with 8% bovine serum (Lubelska Wytw6rnia Surowic i
of the present work was to test the first possibility by comparing the sensitivity of LY-R and LY-S cells and isolated enzymes to topoisomerase II inhibitors. Materials and methods
176
Szczepionek, Lublin, Poland). Cells were maintained in the logarithmic growth phase. Drug solutions were added to 5-ml suspension cultures to obtain the indicated final concentrations. Cultures were kept for 48 h in a CO2 incubator at 37°C and afterwards, cells were counted in a Buerker haemocytometer to determine relative cell numbers (cf. Budzicka et al., 1981). Results were from at least 3 experiments, mean values + standard errors.
Preparation of nuclear extracts Nuclei were isolated according to Pommier et al. (1982). Nuclei were extracted with 0.35 M NaC1 according to Estey et al. (1983). Topoisomerase H assay The topoisomerase II assay determined the conversion of topologically knotted phage P4 DNA to the unknotted topoisomer according to Liu and Davis (1981). The activity was related to
1.01~1
1.000 i
B
~ 0.11~-
~ 0.100
0.010-
0.010 0.000
I O. 1 O0
I 0.200
0.006
0.000
CONCENTRATION OF V P - 1 6 (/~M)
I
I
1.000
2.000
CONCENTRATION OF ADRIAMYCIN (/aM)
1.000~
C
w m
3 Z
0.100-
d
°
o
I 0.010.
0.001 0.000
a 0.100
I 0.200
:
:
0.,300
0.400
CONCENTRATION OF MITOXANTRONE O,I,M) Fig. 1. Drug-induced cytotoxicity in LY-R (©) and LY-S (o) cells: A. VP-16; B. adriamycin; C. mitoxantrone. Cells were exposed to increasing concentrations of drugs for 48 h and then counted as described in the Methods section. Mean values + standard error. The significance levels for the differences between LY-R and LY-S cells are: VP-16 P < 0.05; adriamycin P < 0.01; mitoxantrone P < 0.1 (Student's paired t-test).
177
DNA content in the nuclei determined by OD260 measurements made in 1% SDS, 10 mM EDTA (pH 7.5). One unit unknotted 50% of the substrate DNA after 30 rain at 30°C. Inhibitors were mixed with the enzyme in the assay cocktail in the absence of the substrate and incubated for 10 min at 30°C. Electrophoresis DNA electrophoresis was performed in 0.75% agarose, 2 mM EDTA, 40 mM Tris-acetate (pH 7.8) at 1 V / c m according to Maniatis et al. (1982).
unknotting assay. To quantify the assay, we kept the level of P4 DNA significantly exceeding the enzyme concentration and ATP at the optimal
A. 1
2
3
4
5
6
7
8
Results Susceptibility to topoisomerase H inhibitors at the cellular level For inhibitor studies we chose VP-16 and two intercalating inhibitors of topoisomerase II not tested by Evans et al. (1989): adriamycin and mitoxantrone. To determine the susceptibility to drugs at the cellular level we used a growth test instead of the colony-forming ability test performed by Evans et al. (1989). In the growth test cells were cultured with drugs for 48 h whereas only a 1-h treatment was performed in the colony-forming ability test. Despite this, we found, like those authors, that LY-S ceils were more sensitive than LY-R cells to the cytotoxic effects of VP-16 (Fig. 1A). We also observed a similar but more pronounced effect using adriamycin (Fig. 1B). On the other hand, no significant difference was found between the susceptibility of the two LY strains to mitoxantrone (Fig. 1C). In vitro sensitivity of topoisomerase H to inhibitors To study the sensitivity of topoisomerase II to inhibitors we used a highly specific P4 DNA
Fig. 2. Inhibition of topoisomerase II activity in LY-R and LY-S cells by VP-16. The activity was determined in the nuclear extract prepared as described in the Methods section by the ATP-dependent unknotting of phage P4 DNA in the presence of the indicated concentrations of VP-16. The concentrations of VP-16 were: lanes 1 and 6, 0 t~M; lanes 2 and 7, 7/zM; lanes 3 and 8, 15/xM; lanes 4 and 9, 30/zM; lane 5, control P4 DNA. Electrophorograms (A) were photographed and the amount of unknotted DNA was determined densitometrically (B).
B.
1 O0
m
60-
40, 20. 0
|
0
w
w
10 20 30 CONCENTRATION OF"VP- 16 0~1)
9
178 concentration of 1 mM (Hofmann et al., 1990). Under these conditions the reaction was linear throughout 30 rain. An example of the estimation of the effective concentration of the inhibitor is shown in Fig. 2. The concentration of VP-16 increased by a factor of 2 or 2.5. Scanning of photographs of gels allowed us to determine the concentration of the drug at which 50% of D N A was transformed into an unknotted form. A comparison of the two enzymes indicated that they were identically sensitive to specific inhibitors. The estimated ICs0 values were: 0.42 + 0.08 (for mitoxantrone), 1.2 + 0.3 (for adriamycin) and 10.0 _+ 1.9/xM (for VP16) (mean _+ SE, n = 5). These values are close to those found for the same inhibitors acting on different mammalian type II topoisomerases (Drake et al., 1989; H o f m a n n et al., 1990; Zwelling et al., 1991).
Discussion Topoisomerase II is a target of numerous anticancer drugs which trap the enzyme in the complex with DNA, eventually leading to D N A fragmentation and to cell death (review: Liu, 1989). A number of cell lines have been identified as resistant to topoisomerase II inhibitors. Two main reasons for this have been revealed: a reduced content of topoisomerase II in the cell (Davies et al., 1988; Deffie et al., 1989; Fernandes et al., 1990; Jong et al., 1990) and an altered form of topoisomerase II, resistant to inhibitors (Sullivan et al., 1989; Zwelling et al., 1989). A question raised by us was whether either of the above reasons could be responsible for the higher susceptibility of LY-S cells to inhibitors of topoisomerase II as compared to LY-R cells. In a previous work (Kowalska-Loth et al., submitted) we found that the total unknotting activity measured in nuclear extract was very close for LY-R and LY-S cells, thus pointing to a similar expression of topoisomerase II in both strains. The results of this report indicate a similar sensitivity of enzymes from the two cell lines to topoisomerase II inhibitors. This is true for the non-intercalative VP-16, as well as for the intercalative adriamycin, and also for mitoxantrone which affects more
steps of the topoisomerase II cycle than other inhibitors (Zwelling et al., 1991). The similar effects of these very different inhibitors strongly suggest that the molecular structure of topoisomerase II in LY-S cells is not altered as compared to that of the enzyme from the parental LY-R subline. Despite the above similarities found for topoisomerase II from the two sublines, the results of experiments performed at the cellular level confirm the observation of Evans et al. (1989) that LY-S cells are more susceptible to inhibitors of topoisomerase II than LY-R cells, also in the case of prolonged drug treatment. However, we found that differences between LY-R and LY-S cells in the susceptibility to inhibitors depended on the inhibitor used and, for example, no significant difference was observed for mitoxantrone. This phenomenon could be explained by preferential interactions of different classes of inhibitors at genomic sites differing in importance for cell proliferation (Gewirtz, 1991). Moreover, as mentioned previously, the steps of the topoisomerase II cycle that are affected by mitoxantrone have been found not to overlap with those inhibited by other intercalators or VP-16 (Zwelling et al., 1991). Finally, adriamycin and mitoxantrone not only target on topoisomerase II but also act on plasma membranes (Tritton and Yee, 1982) and may damage D N A due to free radical production (Basra et al., 1985). A question presents itself as to what is the reason for the higher susceptibility of LY-S cells to inhibitors of topoisomerase I! observed both by Evans et al. (1989) and in this work. Evans et al. (1989) have considered as possible reasons an altered topoisomerase II or deficiency in a reaction which is necessary for the repair of D N A double-strand breaks in LY-S cells. The results of our previous (Kowalska-Loth et al., submitted) and the present work exclude the presence of altered topoisomerase II in LY-S cells. Therefore, the latter possibility remains. It is worth noting that an increased susceptibility to m - A M S A was recently found for Chinese hamster ovary xrs-6 cells which, like LY-S cells, were more sensitive to X-radiation than the wild-type cells (Warters et al., 1991). The increased susceptibility was not accompanied by a
179
change in topoisomerase II activity (Warters et al., 1991). Acknowledgements We thank Dr L.F. Liu for the gift of P4 phage and bacterial hosts and Mrs Barbara Pilich for technical assistance. References Alexander, P., and Z.B. Mikulski (1961) Mouse lymphoma cells with different radiosensitivities, Nature, 192, 572-573. Basra, J., C.R. Wolf, J.R. Brown and L.H. Patterson (1985) Evidence for human liver mediated free-radical formation by doxorubicin and mitoxantrone, Anticancer Drug Design, 1, 45-52. Budzicka, E., E. Niepokojczycka and I. Szumiel (1981) Action of misonidazole on L5178Y-R and L5178Y-S cells. I. Comparison with antimycin A and potassium cyanide under aerobic conditions, Neoplasma, 28, 423-433. Davies, S.M., C.N. Robson, S.L. Davies and I.D. Hickson (1988) Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins, J. Biol. Chem., 263, 17724-17729. Deffie, A.M., J.K. Batra and G.J. Goldenberg (1989) Direct correlation between DNA topoisomerase II activity and cytotoxicity in adriamycin-sensitive and -resistant P388 leukemia cell lines, Cancer Res., 49, 58-62. Drake, F.H., G.A. Hofmann, H.B. Bartus, M.R. Mattern, S.T. Crooke and C.K. Mirabelli (1989) Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II, Biochemistry, 28, 8154-8160. Estey, E.H., L. Silberman, M. Beran, B.S. Andersson and L,A. Zwelling (1983) The interaction between nuclear topoisomerase II activity from human leukemia cells, exogenous DNA and 4'-(9-acridinylamino)-methanesulfonem-aniside (m-AMSA) or 4-(4,6-O-ethydiene-/3-o-glucopyranoside) (VP-16) indicates the sensitivity of the cells to the drugs, Biochem. Biophys. Res. Commun., 144, 787-793. Evans, H.H., M. Ricanati, M. Horng and J. Mencl (1989) Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains, Mutation Res., 217, 53-63. Fernandes, D.J., M.K. Danks and W.T. Beck (1990) Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA, Biochemistry, 29, 4235-4241.
Gewirtz, D.A. (1991) Does bulk damage to DNA explain the cytostatic and cytotoxic effects of topoisomerase II inhibitors?, Biochem. Pharmacol., 42, 2253-2258. Hofmann, G.A., C.K. Mirabelli and F.H. Drake (1990) Quantitative adaptation of the bacteriophage P4 DNA unknotting assay for the use in the biochemical and pharmacological characterization of topoisomerase II, Anticancer Drug Design, 5, 273-282. Jong, S., G. Zijlstra, E.G.E. Vries and N.H. Mulder (1990) Reduced DNA topoisomerase II activity and drug-induced DNA cleavage activity in an adriamycin-resistant human small cell lung carcinoma cell line, Cancer Res., 50, 304309. Kowalska-Loth, B., K. Staron, I. Buraczewska, I. Szumiel, M. Kapiszewska and C.S. Lange (1992) Reduced sensitivity to camptothecin of topoisomerase I from a L5178Y mouse lymphoma subline sensitive to X-radiation, submitted. Liu, L.F. (1989) DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem., 58, 351-375. Liu, L.F., and J.L. Davis (1981) Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases, Nucleic Acids Res., 9, 3979-3989. Maniatis, T., E. Fritsch and J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Pommier, Y., D. Kerrigan, R. Schwartz and LA. Zwelling (1982) The formation and resealing of intercalator-induced DNA strand breaks in isolated L1210 nuclei, Biochem. Biophys. Res. Commun., 107, 576-583. Sullivan, D.M., M.D. Latham, T.C. Rowe and W.E. Ross (1989) Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line, Biochemistry, 28, 5680-5687. Tritton, T.R., and G. Yee (1982) The anticancer agent adriamycin can be actively cytotoxic without entering cells, Science, 217, 248-250. Warters, R.L., B.W. Lyons, T.M. Li and D.J. Chen (1991) Topoisomerase 1I activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line, Mutation Res., 254, 167-174. Zwelling, L.A., M. Hinds, D. Chan, J. Mayes, K.L. Sie, E. Parker, L Silberman, A. Radcliffe, M. Beran and M. Blick (1989) Characterization of an amsacrine-resistant line of human leukemia cells, J. Biol. Chem., 264, 16411-16420. Zwelling, L.A., J. Mayes, M. Hinds, D. Chan, E. Altschuler, B. Carrol, E. Parker, K. Deisseroth, A. Radcliffe, M. Seligman, L. Li and D. Farquhar (1991) Topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions, Biochemistry, 30, 4048-4055.
175
MUT 05193
Sensitivity to inhibitors of type II topoisomerases from mouse L5178Y lymphoma strains that are resistant or sensitive to X-radiation Krzysztof Staron a, Barbara Kowalska-Loth a, Irena Szumiel b and Iwona Buraczewska b a Institute of Biochemistry, Warsaw University, Warsaw, Poland and ~ Institute of Nuclear Chemistry and Technology, Warsaw, Poland (Received 26 March 1992) (Revision received 4 August 1992) (Accepted 6 August 1992)
Keywords: Topoisomerase II inhibitors; L5178Y mouse lymphoma cells
Summary Sensitivity to topoisomerase II inhibitors was tested at the cellular and enzyme level for two strains of mouse L5178Y lymphoma cells: resistant (LY-R) and sensitive (LY-S) to X-radiation. Differences in the susceptibility to inhibitors between LY-R and LY-S cells depended on the inhibitor used and were observed for adriamycin and VP-16, but not for mitoxantrone. On the other hand, isolated enzymes displayed the same sensitivity to all inhibitors tested regardless of the cell line. These results exclude the presence of altered topoisomerase II in LY-S cells as a possible reason for the collateral sensitivity of LY-S cells to X-radiation and topoisomerase II inhibitors.
Two strains of mouse lymphoma L5178Y cells, which differ in their sensitivity to X-radiation (Alexander and Mikulski, t961), have been found to be differently sensitive to topoisomerase II inhibitors: m-AMSA, VP-16 and ellipticine (Evans et al., 1989). Strain LY-S, which is sensitive to X-radiation compared to strain LY-R, is also more sensitive to the above inhibitors of topoisomerase II. The concomitance of radiation sensitivity and susceptibility to topoisomerase II poisons has been suggested to result either (1) from altered topoisomerase II in strain LY-S or (2) from the DNA double-strain break repair deficiency in that strain (Evans et al., 1989). The aim
Materials Drugs used in this work were commercial pharmaceutical preparations: adriamycin (Adriblastina; Farmitalia, Carlo Erba, Milan, Italy), mitoxantrone (Novatron; Cyanamid GmbH, Wolfratshausen, Germany) and VP-16 (Vepesid; Bristol-Myers GmbH, Bergisch-Gladbach, Germany).
Correspondence: Krzysztof Staron, Institute of Biochemistry, Warsaw University, AI. Zwirki i Wigury 93, 02-089 Warsaw, Poland.
Cell culture LY-R and -S cells were cultured in suspension in Fischer's medium supplemented with 8% bovine serum (Lubelska Wytw6rnia Surowic i
of the present work was to test the first possibility by comparing the sensitivity of LY-R and LY-S cells and isolated enzymes to topoisomerase II inhibitors. Materials and methods
176
Szczepionek, Lublin, Poland). Cells were maintained in the logarithmic growth phase. Drug solutions were added to 5-ml suspension cultures to obtain the indicated final concentrations. Cultures were kept for 48 h in a CO2 incubator at 37°C and afterwards, cells were counted in a Buerker haemocytometer to determine relative cell numbers (cf. Budzicka et al., 1981). Results were from at least 3 experiments, mean values + standard errors.
Preparation of nuclear extracts Nuclei were isolated according to Pommier et al. (1982). Nuclei were extracted with 0.35 M NaC1 according to Estey et al. (1983). Topoisomerase H assay The topoisomerase II assay determined the conversion of topologically knotted phage P4 DNA to the unknotted topoisomer according to Liu and Davis (1981). The activity was related to
1.01~1
1.000 i
B
~ 0.11~-
~ 0.100
0.010-
0.010 0.000
I O. 1 O0
I 0.200
0.006
0.000
CONCENTRATION OF V P - 1 6 (/~M)
I
I
1.000
2.000
CONCENTRATION OF ADRIAMYCIN (/aM)
1.000~
C
w m
3 Z
0.100-
d
°
o
I 0.010.
0.001 0.000
a 0.100
I 0.200
:
:
0.,300
0.400
CONCENTRATION OF MITOXANTRONE O,I,M) Fig. 1. Drug-induced cytotoxicity in LY-R (©) and LY-S (o) cells: A. VP-16; B. adriamycin; C. mitoxantrone. Cells were exposed to increasing concentrations of drugs for 48 h and then counted as described in the Methods section. Mean values + standard error. The significance levels for the differences between LY-R and LY-S cells are: VP-16 P < 0.05; adriamycin P < 0.01; mitoxantrone P < 0.1 (Student's paired t-test).
177
DNA content in the nuclei determined by OD260 measurements made in 1% SDS, 10 mM EDTA (pH 7.5). One unit unknotted 50% of the substrate DNA after 30 rain at 30°C. Inhibitors were mixed with the enzyme in the assay cocktail in the absence of the substrate and incubated for 10 min at 30°C. Electrophoresis DNA electrophoresis was performed in 0.75% agarose, 2 mM EDTA, 40 mM Tris-acetate (pH 7.8) at 1 V / c m according to Maniatis et al. (1982).
unknotting assay. To quantify the assay, we kept the level of P4 DNA significantly exceeding the enzyme concentration and ATP at the optimal
A. 1
2
3
4
5
6
7
8
Results Susceptibility to topoisomerase H inhibitors at the cellular level For inhibitor studies we chose VP-16 and two intercalating inhibitors of topoisomerase II not tested by Evans et al. (1989): adriamycin and mitoxantrone. To determine the susceptibility to drugs at the cellular level we used a growth test instead of the colony-forming ability test performed by Evans et al. (1989). In the growth test cells were cultured with drugs for 48 h whereas only a 1-h treatment was performed in the colony-forming ability test. Despite this, we found, like those authors, that LY-S ceils were more sensitive than LY-R cells to the cytotoxic effects of VP-16 (Fig. 1A). We also observed a similar but more pronounced effect using adriamycin (Fig. 1B). On the other hand, no significant difference was found between the susceptibility of the two LY strains to mitoxantrone (Fig. 1C). In vitro sensitivity of topoisomerase H to inhibitors To study the sensitivity of topoisomerase II to inhibitors we used a highly specific P4 DNA
Fig. 2. Inhibition of topoisomerase II activity in LY-R and LY-S cells by VP-16. The activity was determined in the nuclear extract prepared as described in the Methods section by the ATP-dependent unknotting of phage P4 DNA in the presence of the indicated concentrations of VP-16. The concentrations of VP-16 were: lanes 1 and 6, 0 t~M; lanes 2 and 7, 7/zM; lanes 3 and 8, 15/xM; lanes 4 and 9, 30/zM; lane 5, control P4 DNA. Electrophorograms (A) were photographed and the amount of unknotted DNA was determined densitometrically (B).
B.
1 O0
m
60-
40, 20. 0
|
0
w
w
10 20 30 CONCENTRATION OF"VP- 16 0~1)
9
178 concentration of 1 mM (Hofmann et al., 1990). Under these conditions the reaction was linear throughout 30 rain. An example of the estimation of the effective concentration of the inhibitor is shown in Fig. 2. The concentration of VP-16 increased by a factor of 2 or 2.5. Scanning of photographs of gels allowed us to determine the concentration of the drug at which 50% of D N A was transformed into an unknotted form. A comparison of the two enzymes indicated that they were identically sensitive to specific inhibitors. The estimated ICs0 values were: 0.42 + 0.08 (for mitoxantrone), 1.2 + 0.3 (for adriamycin) and 10.0 _+ 1.9/xM (for VP16) (mean _+ SE, n = 5). These values are close to those found for the same inhibitors acting on different mammalian type II topoisomerases (Drake et al., 1989; H o f m a n n et al., 1990; Zwelling et al., 1991).
Discussion Topoisomerase II is a target of numerous anticancer drugs which trap the enzyme in the complex with DNA, eventually leading to D N A fragmentation and to cell death (review: Liu, 1989). A number of cell lines have been identified as resistant to topoisomerase II inhibitors. Two main reasons for this have been revealed: a reduced content of topoisomerase II in the cell (Davies et al., 1988; Deffie et al., 1989; Fernandes et al., 1990; Jong et al., 1990) and an altered form of topoisomerase II, resistant to inhibitors (Sullivan et al., 1989; Zwelling et al., 1989). A question raised by us was whether either of the above reasons could be responsible for the higher susceptibility of LY-S cells to inhibitors of topoisomerase II as compared to LY-R cells. In a previous work (Kowalska-Loth et al., submitted) we found that the total unknotting activity measured in nuclear extract was very close for LY-R and LY-S cells, thus pointing to a similar expression of topoisomerase II in both strains. The results of this report indicate a similar sensitivity of enzymes from the two cell lines to topoisomerase II inhibitors. This is true for the non-intercalative VP-16, as well as for the intercalative adriamycin, and also for mitoxantrone which affects more
steps of the topoisomerase II cycle than other inhibitors (Zwelling et al., 1991). The similar effects of these very different inhibitors strongly suggest that the molecular structure of topoisomerase II in LY-S cells is not altered as compared to that of the enzyme from the parental LY-R subline. Despite the above similarities found for topoisomerase II from the two sublines, the results of experiments performed at the cellular level confirm the observation of Evans et al. (1989) that LY-S cells are more susceptible to inhibitors of topoisomerase II than LY-R cells, also in the case of prolonged drug treatment. However, we found that differences between LY-R and LY-S cells in the susceptibility to inhibitors depended on the inhibitor used and, for example, no significant difference was observed for mitoxantrone. This phenomenon could be explained by preferential interactions of different classes of inhibitors at genomic sites differing in importance for cell proliferation (Gewirtz, 1991). Moreover, as mentioned previously, the steps of the topoisomerase II cycle that are affected by mitoxantrone have been found not to overlap with those inhibited by other intercalators or VP-16 (Zwelling et al., 1991). Finally, adriamycin and mitoxantrone not only target on topoisomerase II but also act on plasma membranes (Tritton and Yee, 1982) and may damage D N A due to free radical production (Basra et al., 1985). A question presents itself as to what is the reason for the higher susceptibility of LY-S cells to inhibitors of topoisomerase I! observed both by Evans et al. (1989) and in this work. Evans et al. (1989) have considered as possible reasons an altered topoisomerase II or deficiency in a reaction which is necessary for the repair of D N A double-strand breaks in LY-S cells. The results of our previous (Kowalska-Loth et al., submitted) and the present work exclude the presence of altered topoisomerase II in LY-S cells. Therefore, the latter possibility remains. It is worth noting that an increased susceptibility to m - A M S A was recently found for Chinese hamster ovary xrs-6 cells which, like LY-S cells, were more sensitive to X-radiation than the wild-type cells (Warters et al., 1991). The increased susceptibility was not accompanied by a
179
change in topoisomerase II activity (Warters et al., 1991). Acknowledgements We thank Dr L.F. Liu for the gift of P4 phage and bacterial hosts and Mrs Barbara Pilich for technical assistance. References Alexander, P., and Z.B. Mikulski (1961) Mouse lymphoma cells with different radiosensitivities, Nature, 192, 572-573. Basra, J., C.R. Wolf, J.R. Brown and L.H. Patterson (1985) Evidence for human liver mediated free-radical formation by doxorubicin and mitoxantrone, Anticancer Drug Design, 1, 45-52. Budzicka, E., E. Niepokojczycka and I. Szumiel (1981) Action of misonidazole on L5178Y-R and L5178Y-S cells. I. Comparison with antimycin A and potassium cyanide under aerobic conditions, Neoplasma, 28, 423-433. Davies, S.M., C.N. Robson, S.L. Davies and I.D. Hickson (1988) Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins, J. Biol. Chem., 263, 17724-17729. Deffie, A.M., J.K. Batra and G.J. Goldenberg (1989) Direct correlation between DNA topoisomerase II activity and cytotoxicity in adriamycin-sensitive and -resistant P388 leukemia cell lines, Cancer Res., 49, 58-62. Drake, F.H., G.A. Hofmann, H.B. Bartus, M.R. Mattern, S.T. Crooke and C.K. Mirabelli (1989) Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II, Biochemistry, 28, 8154-8160. Estey, E.H., L. Silberman, M. Beran, B.S. Andersson and L,A. Zwelling (1983) The interaction between nuclear topoisomerase II activity from human leukemia cells, exogenous DNA and 4'-(9-acridinylamino)-methanesulfonem-aniside (m-AMSA) or 4-(4,6-O-ethydiene-/3-o-glucopyranoside) (VP-16) indicates the sensitivity of the cells to the drugs, Biochem. Biophys. Res. Commun., 144, 787-793. Evans, H.H., M. Ricanati, M. Horng and J. Mencl (1989) Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains, Mutation Res., 217, 53-63. Fernandes, D.J., M.K. Danks and W.T. Beck (1990) Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA, Biochemistry, 29, 4235-4241.
Gewirtz, D.A. (1991) Does bulk damage to DNA explain the cytostatic and cytotoxic effects of topoisomerase II inhibitors?, Biochem. Pharmacol., 42, 2253-2258. Hofmann, G.A., C.K. Mirabelli and F.H. Drake (1990) Quantitative adaptation of the bacteriophage P4 DNA unknotting assay for the use in the biochemical and pharmacological characterization of topoisomerase II, Anticancer Drug Design, 5, 273-282. Jong, S., G. Zijlstra, E.G.E. Vries and N.H. Mulder (1990) Reduced DNA topoisomerase II activity and drug-induced DNA cleavage activity in an adriamycin-resistant human small cell lung carcinoma cell line, Cancer Res., 50, 304309. Kowalska-Loth, B., K. Staron, I. Buraczewska, I. Szumiel, M. Kapiszewska and C.S. Lange (1992) Reduced sensitivity to camptothecin of topoisomerase I from a L5178Y mouse lymphoma subline sensitive to X-radiation, submitted. Liu, L.F. (1989) DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem., 58, 351-375. Liu, L.F., and J.L. Davis (1981) Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases, Nucleic Acids Res., 9, 3979-3989. Maniatis, T., E. Fritsch and J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Pommier, Y., D. Kerrigan, R. Schwartz and LA. Zwelling (1982) The formation and resealing of intercalator-induced DNA strand breaks in isolated L1210 nuclei, Biochem. Biophys. Res. Commun., 107, 576-583. Sullivan, D.M., M.D. Latham, T.C. Rowe and W.E. Ross (1989) Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line, Biochemistry, 28, 5680-5687. Tritton, T.R., and G. Yee (1982) The anticancer agent adriamycin can be actively cytotoxic without entering cells, Science, 217, 248-250. Warters, R.L., B.W. Lyons, T.M. Li and D.J. Chen (1991) Topoisomerase 1I activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line, Mutation Res., 254, 167-174. Zwelling, L.A., M. Hinds, D. Chan, J. Mayes, K.L. Sie, E. Parker, L Silberman, A. Radcliffe, M. Beran and M. Blick (1989) Characterization of an amsacrine-resistant line of human leukemia cells, J. Biol. Chem., 264, 16411-16420. Zwelling, L.A., J. Mayes, M. Hinds, D. Chan, E. Altschuler, B. Carrol, E. Parker, K. Deisseroth, A. Radcliffe, M. Seligman, L. Li and D. Farquhar (1991) Topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions, Biochemistry, 30, 4048-4055.