BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 176, No. 2, 1991
Pages 690-697
April 30, 1991
DNA TOPOISOMERASE lI FROM MAMMALIAN MITOCHONDRIA IS INHIBITED BY T H E ANTITUMOR DRUGS, m-AMSA AND VM-26 Jia-Hwei Lin and Frank J. Castora* Laboratory of Molecular Biochemistry Department of Biochemistry Eastern Virginia Medical School Norfolk, VA 23507-1696 Received ~ r c h
14, 1991
A type II DNA topoisomerase has been partially purified from calf thymus mitochondria by a combination of differential centrifugation and column chromatography. The mitochondrial enzyme was inhibited by amsacrine (m-AMSA) slightly at 0.5 #M, significantly at 5.0 juM, and completely at 50 juM. A similar profile was obtained with teniposide (VM-26) although the latter drug was not quite as potent an inhibitor as the former. P4 unknotting assays of the purified nuclear type II topoisomerase in the presence of m-AMSA and VM-26 indicated that the mitochondrial and nuclear enzymes behaved similarly, although the mitochondrial enzyme appeared to be inhibited more strongly. © 1991 A c a d e m i c
Press,
Inc.
Mammalian mitochondrial DNA is a circular molecule of approximately 16,500 base pairs.
Replication and transcription of such a circular DNA will generate topological
tension which must be relieved to allow these processes to continue. DNA topoisomerases interconvert various topological forms of DNA that arise during DNA replication and transcription and by so doing they ease the developing topological stress (for reviews see 1-3). Type I DNA topoisomerases make and reseal single strand breaks while type H enzymes make and reseal double stranded breaks in the DNA. Our lab has purified a type I DNA topoisomerase from calf thymus mitochondria (4). A type II DNA topoisomerase activity had also been observed in mitochondria of rat liver (5) and human leukemia (HALL) cells (6).
However, due to the limitation of the
obtainable material, no mammalian mitochondrial DNA topoisomerase II has yet been extensively purified. Melendy and Ray (7) have recently purified a mitochondrial type II * To whom correspondence should be addressed. Abbreviations - KPi, potassium phosphate; m-AMSA, amsacrine or (4'-(9-acridinylamino)-methanesulfon-m-anisidine; mtDNA, mitochondrial DNA; PEG, polyethyleneglycol; VM-26, teniposide. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
690
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA topoisomerase from the kinetoplasts of the Trypanosomatid Crithidia fasciculata. No equivalent level of purification has been reported for a mammalian mitochondrial enzyme. Recently, a number of in vitro and in vivo studies have shown that the DNA topoisomerases are targets of a number of anti-tumor drugs.
Camptothecin, a plant
alkaloid, selectively inhibits eukaryotic DNA topoisomerase I (8). DNA topoisomerase II is targeted by acridines, anthocycline, and epipodophyllotoxins (for review, see 9). Thus far, the effects of these drugs have been observed with nuclear topoisomerases. However, the DNA topoisomerase II from the C. fasciculata kinetoplasts was inhibited in vitro by amsacrine (m-AMSA), etoposide (VP16-213) and teniposide (VM-26) (7). Shapiro et al. (10) have demonstrated in vivo drug-promoted cleavage of kinetoplast DNA minicircles, but no reports of effects on mammalian organelle topoisomerases have yet been published. We report here the isolation of DNA topoisomerase II from calf thymus mitochondria and the sensitivity of this mitochondrial enzyme to the antitumor drugs m-AMSA and VM-26.
MATERIALS AND METHODS DNA topoisomerase I assay: Standard DNA topoisomerase I activity was measured by the extent of relaxation of supercoiled pUC19 DNA as described previously (4). One unit of activity relaxes 50% of the substrate DNA at 30°C in 30 min. DNA topoisomerase II assay: P4 unknotting assay was used to measure DNA topoisomerase II activity as described by Liu et al. (11). One unit of activity converts 50% of the knotted DNA into monomer at 30°C in 30 rain. Protein determination: Protein was measured by biuret analysis (12) at the early stages of purification and by the method of Bradford (13) after the column chromatography. Bovine serum albumin was used as standard protein in either procedure. Isolation of mitoehondria and mitoplasts: The mitochondria, mitoplasts and the inner membranes were isolated essentially as described previously (4) except that a sucrose/Percoll gradient was used to band purified mitochondria in a 20 min centrifugation. Isolation of DNA topoisomerase II from the mitochondrial inner membranes: All steps after the lysis of the purified mitochondria contained a protease inhibitor cocktail as described in (4). All potassium phosphate (KPi) buffers were pH 7.3. The inner membranes were solubilized with Triton X 100 in 150 mM KPi/1.0 M NaC1/20% glycerol and the mitochondrial DNA was removed by polyethylene glycol (PEG) precipitation. After centrifugation, the PEG supernatant was diluted with 20% glycerol to 0.33 M NaC1 and loaded on a phosphocellulose column equilibrated with 50 mM KPi/0.33 M NaC1/20% glycerol. After washing with three bed volumes of starting buffer, the enzyme was eluted with 50 mM KPi/0.8 M NaC1/20% glycerol. Active fractions were combined and loaded directly on a hydroxylapatite column equilibrated in 50 mM KPi/0.8 M NaC1/20% glycerol After washing with this starting buffer and three bed volumes of 0.2 M KPi/20% glyceroi, the enzyme was eluted with a single step of 0.5 M KPi/20% glycerol. The active fractions were dialyzed into storage buffer (0.5 M KPi/50% glycerol) and kept at -20°C. Marker enzyme assays: Glucose-6-phosphate dehydrogenase, sulfite oxidase, succinic dehydrogenase, amine oxidase and DNA polymerases alpha and gamma were assayed 691
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
essentially as described in (4). The amount of polymerase beta and gamma activity that contributed to the alpha polymerase assay was determined by performing an alpha polymerase assay in the presence of aphidicolin (14). This corrected activity, pol alpha', was calculated from the following equation pol alpha' -- pol alphatot,l - (pol alpha plus aphidicolin) and was used to calculate the ratio of gamma to alpha polymerase in our final preparation. RESULTS AND DISCUSSION Isolation of mitochondrial topoisomerase II from calf thymus. Mitochondria were prepared from calf thymus essentially as described previously (4) by a series of differential and sucrose/Percoll density gradient centrifugations. After stripping away the mitochondrial outer membrane with digitonin (15), mitochondrial inner membranes were isolated by three cycles of freezing and thawing as described earlier (4). Proteins were solubilized by treatment with Triton X 100 and the lysate was cleared of mitochondrial DNA (mtDNA) by polyethyleneglycol (PEG)
precipitation
in high
salt.
After fractionation
by
phosphocellulose and hydroxylapatite chromatography, the partially purified mitochondrial topoisomerase II was dialyzed into storage buffer where it has been stable at -20°C for several months. As seen in Table I, the mitochondrial topoisomerase II was purified over 3000-fold compared to purified mitochondria. Since mitochondrial proteins make up only approximately 25% of the total cellular proteins, this represents more than a 12,000-fold purification relative to whole cell protein. Because nuclease activity is present in early fractions, it was impossible to quantitate the mitochondrial topoisomerase u_nknotting activity until after the supernatant from the PEG precipitation was fractionated on phosphocellulose. The overall yield was 9% based on the assumption that there was a constant amount of enzyme activity from crude mitochondria to the phosphocellulose chromatography. Throughout the isolation procedure activities for a variety of marker enzymes were determined as previously described (4). As expected, glucose-6-phosphate dehydrogenase activity decreased to non-detectable levels in the PEG-supernatant. Likewise, sulfite oxidase and amine oxidase decreased dramatically while succinic dehydrogenase levels were high in the PEG-supernatant which was derived from the inner membranes. An important indicator of the purity of the mitochondrial preparation and the mitochondrial origin of the topoisomerase isolated is the ratio of DNA polymerase gamma (the mitochondrial matrix enzyme responsible for mtDNA replication) to the DNA polymerase alpha (the nuclear polymerase responsible for chromosomal DNA replication). 692
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I SUMMARY OF THE PURIFICATION OF DNA TOPOISOMERASE II FROM CALF THYMUS MITOCHONDRIA STEP
FRACTION
VOLUME TOTAL SPECIFIC PURIF. YIELD PROTEIN ACTIVITY ACTIVITY (mL) (mg) (UNITS) (U/rag) (FOLD) (%)
I CRUDE MITOCHONDRIA 210
1316
11500
8.7
1
100
965
11500
11.9
1
100
40
197.5
11500
58.2
4.9
100
120
87.7
11500
131
11
100
11.5
0.805 11500 14286
1200
100
VI HYDROXYLAPATITE COLUMN FRACTION
0.66
0.085 1320 15529
1305
11.5
VII DIALYZED FRACTION
0.26
0.028 1040 37142
3121
9.0
II PURE MITOCHONDRIA 1II MITOPLAST IV DILUTED PEG-SUPE V PHOSPHOCELLULOSE COLUMN COMBINED
95
Starting material was 945 grams of fresh calf thymus gland. Preparation of various intermediate fractions and column chromatograaphy was as described in MATERIALS AND METHODS.
A ratio of at least 30:1 gamma:alpha polymerase has been reported in the past as a marker for mitochondrial enzymes (4,16,17). Because D N A polymerases g a m m a and beta
are somewhat active under alpha
polymerase assay conditions, we used aphidicolin, a selective inhibitor of alpha polymerase (14), to determine the amount of contributing beta and gamma activity in our assay. As seen in Table II, the ratio of g a m m a to alpha polymerase increases in each purification step and in the PEG-supernatant it is almost 80:1. This is in accord with previous reports (4,16,17) and, together with the other marker enzyme values, establishes the mitochondrial origin of our enzyme.
The mitochondrial topoisomerase unknots D N A in an ATP-dependent reaction. One of the features of eukaryotic type II topoisomerases is their ability to remove supercoils and/or knots from D N A in the presence but not the absence of ATP. Although relaxation of supercoils in circular D N A can be achieved by either a type I or II topoisomerase, the unknotting of phage P4 D N A is catalyzed by type II but not type I topoisomerases (11). Therefore, this latter activity is considered indicative of a type II topoisomerase. Fig. 1 shows the results of treating knotted P4 DNA with the purified mitochondrial enzyme in 693
V o l . 176, N o . 2, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II DNA POLYMERASE
Fraction
ACTIVITIES
IN M I T O C H O N D R I A L
Polymerase Actvity" alpha
Crude mito
FRACTIONS
Ratio
gamma
gamma/alpha
67
287
4.3/1
Mitoplast
4
72
18/l
PEG supe
1.3
101
78/1
a DNA polymerase activity is shown as units determined as described in Materials and Methods.
the p r e s e n c e and a b s e n c e of A T P . Without a d d e d A T P (lane b) the substrate D N A is u n c h a n g e d c o m p a r e d to u n t r e a t e d control (lane a) which a p p e a r s as a diffuse s m e a r u p o n a g a r o s e gel electrophoresis. With the a d d i t i o n of 1 m M A T P , however, the D N A is extensively u n k n o t t e d a n d a p p e a r s as an intense, slower migrating band (lanes c-f). As the m i t o c h o n d r i a l e n z y m e is diluted this unknotting activity c o r r e s p o n d i n g l y decreases.
abcdef
M-
abc
defg
hi
j k I rn
L-
(D
®
Fig. 1. P4 unknotting assay of mitochondrial DNA topoisomerase II in the presence or absence of ATP. Lane a, P4 DNA only; lane b, plus enzyme minus ATP; lane c-f contain serial diluted enzyme plus 1 mM ATP; lane c, 1 uL; lane d, 1/2 uL; lane e, 1/4 uL; lane f, 1/8 uL. DNA bands labelled M and L are monomer and linear P4 DNA, respectively. Fig. 2. P4 unknotting assay of mitochondriat topoisomerase II in the presence or absence of antitumor drugs. Lane a, P4 DNA only; lane b, P4 DNA plus enzyme plus 1 mM ATP; lane c, same as lane b minus ATP. Lane d-g, same as lane b plus the indicated amounts of m-AMSA: lane d, 0.5 uM; lane e, 5 uM; lane f, 50 uM; lane g, 150 uM. Lane h, same as lane g minus enzyme. Lane i-l, same as lane b plus the indicated amounts of VM-26; lane i, 0.5 uM; lane j, 5 uM; lane k, 50 uM; lane 1, 150 uM. Lane m, same as lane 1 minus enzyme. 694
V o I . 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Response of the mitochondrial topoisomerase to the antitumor drugs, mAMSA and VM-26.
Recently, it has come to light that the type I and II topoisomerases are the
intracellular targets of a number of antitumor drugs (9). For example, the plant alkaloid, camptothecin, has been shown to selectively inhibit the nuclear type I topoisomerase (8). The anticaneer drugs, mAMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidine) and VM26, have been shown to inhibit the nuclear type II topoisomerase (18,19). Since these initial reports, a large number of studies have addressed the biochemical, biological, and physiological consequences of the drug-induced inhibition of topoisomerases. These many studies have concerned, almost exclusively, effects on the nuclear enzymes. There is little known of the response of mitochondrial topoisomerases to these antitumor drugs. We therefore determined the sensitivity of our purified mitochondrial topoisomerase II to two anti-neoplastic drugs, mAMSA and VM-26. Figure 2 shows the results of topoisomerase II assays in the presence and absence of mAMSA and VM-26. There is significant inhibition by as little as 0.5 uM mAMSA and essentially complete inhibition by 50 uM mAMSA (compare lanes d and f to lane b). Likewise, VM-26 inhibits the mitochondrial enzyme significantly at low concentrations with close to complete inhibition by 50 uM (lanes i and k). Both the intercalating drug (mAMSA) and the non-intercalating drug (VM-26) can inhibit the mitochondrial topoisomerase II with a similar profile although it seems that mAMSA may be a slightly more potent inhibitor. The mitochondrial topoisomerase II is more sensitive to mAMSA and VM-26 than the nuclear enzyme. Considering the many previous drug studies and our own results, it is clear that these anti-neoplastic drugs can inhibit all the cellular topoisomerases, those in the
2.0
!\
5
:\ \ \ \ 7"\
~_ 1.s >
I--O
Vol. 176, No. 2, 1991
Pages 690-697
April 30, 1991
DNA TOPOISOMERASE lI FROM MAMMALIAN MITOCHONDRIA IS INHIBITED BY T H E ANTITUMOR DRUGS, m-AMSA AND VM-26 Jia-Hwei Lin and Frank J. Castora* Laboratory of Molecular Biochemistry Department of Biochemistry Eastern Virginia Medical School Norfolk, VA 23507-1696 Received ~ r c h
14, 1991
A type II DNA topoisomerase has been partially purified from calf thymus mitochondria by a combination of differential centrifugation and column chromatography. The mitochondrial enzyme was inhibited by amsacrine (m-AMSA) slightly at 0.5 #M, significantly at 5.0 juM, and completely at 50 juM. A similar profile was obtained with teniposide (VM-26) although the latter drug was not quite as potent an inhibitor as the former. P4 unknotting assays of the purified nuclear type II topoisomerase in the presence of m-AMSA and VM-26 indicated that the mitochondrial and nuclear enzymes behaved similarly, although the mitochondrial enzyme appeared to be inhibited more strongly. © 1991 A c a d e m i c
Press,
Inc.
Mammalian mitochondrial DNA is a circular molecule of approximately 16,500 base pairs.
Replication and transcription of such a circular DNA will generate topological
tension which must be relieved to allow these processes to continue. DNA topoisomerases interconvert various topological forms of DNA that arise during DNA replication and transcription and by so doing they ease the developing topological stress (for reviews see 1-3). Type I DNA topoisomerases make and reseal single strand breaks while type H enzymes make and reseal double stranded breaks in the DNA. Our lab has purified a type I DNA topoisomerase from calf thymus mitochondria (4). A type II DNA topoisomerase activity had also been observed in mitochondria of rat liver (5) and human leukemia (HALL) cells (6).
However, due to the limitation of the
obtainable material, no mammalian mitochondrial DNA topoisomerase II has yet been extensively purified. Melendy and Ray (7) have recently purified a mitochondrial type II * To whom correspondence should be addressed. Abbreviations - KPi, potassium phosphate; m-AMSA, amsacrine or (4'-(9-acridinylamino)-methanesulfon-m-anisidine; mtDNA, mitochondrial DNA; PEG, polyethyleneglycol; VM-26, teniposide. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
690
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DNA topoisomerase from the kinetoplasts of the Trypanosomatid Crithidia fasciculata. No equivalent level of purification has been reported for a mammalian mitochondrial enzyme. Recently, a number of in vitro and in vivo studies have shown that the DNA topoisomerases are targets of a number of anti-tumor drugs.
Camptothecin, a plant
alkaloid, selectively inhibits eukaryotic DNA topoisomerase I (8). DNA topoisomerase II is targeted by acridines, anthocycline, and epipodophyllotoxins (for review, see 9). Thus far, the effects of these drugs have been observed with nuclear topoisomerases. However, the DNA topoisomerase II from the C. fasciculata kinetoplasts was inhibited in vitro by amsacrine (m-AMSA), etoposide (VP16-213) and teniposide (VM-26) (7). Shapiro et al. (10) have demonstrated in vivo drug-promoted cleavage of kinetoplast DNA minicircles, but no reports of effects on mammalian organelle topoisomerases have yet been published. We report here the isolation of DNA topoisomerase II from calf thymus mitochondria and the sensitivity of this mitochondrial enzyme to the antitumor drugs m-AMSA and VM-26.
MATERIALS AND METHODS DNA topoisomerase I assay: Standard DNA topoisomerase I activity was measured by the extent of relaxation of supercoiled pUC19 DNA as described previously (4). One unit of activity relaxes 50% of the substrate DNA at 30°C in 30 min. DNA topoisomerase II assay: P4 unknotting assay was used to measure DNA topoisomerase II activity as described by Liu et al. (11). One unit of activity converts 50% of the knotted DNA into monomer at 30°C in 30 rain. Protein determination: Protein was measured by biuret analysis (12) at the early stages of purification and by the method of Bradford (13) after the column chromatography. Bovine serum albumin was used as standard protein in either procedure. Isolation of mitoehondria and mitoplasts: The mitochondria, mitoplasts and the inner membranes were isolated essentially as described previously (4) except that a sucrose/Percoll gradient was used to band purified mitochondria in a 20 min centrifugation. Isolation of DNA topoisomerase II from the mitochondrial inner membranes: All steps after the lysis of the purified mitochondria contained a protease inhibitor cocktail as described in (4). All potassium phosphate (KPi) buffers were pH 7.3. The inner membranes were solubilized with Triton X 100 in 150 mM KPi/1.0 M NaC1/20% glycerol and the mitochondrial DNA was removed by polyethylene glycol (PEG) precipitation. After centrifugation, the PEG supernatant was diluted with 20% glycerol to 0.33 M NaC1 and loaded on a phosphocellulose column equilibrated with 50 mM KPi/0.33 M NaC1/20% glycerol. After washing with three bed volumes of starting buffer, the enzyme was eluted with 50 mM KPi/0.8 M NaC1/20% glycerol. Active fractions were combined and loaded directly on a hydroxylapatite column equilibrated in 50 mM KPi/0.8 M NaC1/20% glycerol After washing with this starting buffer and three bed volumes of 0.2 M KPi/20% glyceroi, the enzyme was eluted with a single step of 0.5 M KPi/20% glycerol. The active fractions were dialyzed into storage buffer (0.5 M KPi/50% glycerol) and kept at -20°C. Marker enzyme assays: Glucose-6-phosphate dehydrogenase, sulfite oxidase, succinic dehydrogenase, amine oxidase and DNA polymerases alpha and gamma were assayed 691
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
essentially as described in (4). The amount of polymerase beta and gamma activity that contributed to the alpha polymerase assay was determined by performing an alpha polymerase assay in the presence of aphidicolin (14). This corrected activity, pol alpha', was calculated from the following equation pol alpha' -- pol alphatot,l - (pol alpha plus aphidicolin) and was used to calculate the ratio of gamma to alpha polymerase in our final preparation. RESULTS AND DISCUSSION Isolation of mitochondrial topoisomerase II from calf thymus. Mitochondria were prepared from calf thymus essentially as described previously (4) by a series of differential and sucrose/Percoll density gradient centrifugations. After stripping away the mitochondrial outer membrane with digitonin (15), mitochondrial inner membranes were isolated by three cycles of freezing and thawing as described earlier (4). Proteins were solubilized by treatment with Triton X 100 and the lysate was cleared of mitochondrial DNA (mtDNA) by polyethyleneglycol (PEG)
precipitation
in high
salt.
After fractionation
by
phosphocellulose and hydroxylapatite chromatography, the partially purified mitochondrial topoisomerase II was dialyzed into storage buffer where it has been stable at -20°C for several months. As seen in Table I, the mitochondrial topoisomerase II was purified over 3000-fold compared to purified mitochondria. Since mitochondrial proteins make up only approximately 25% of the total cellular proteins, this represents more than a 12,000-fold purification relative to whole cell protein. Because nuclease activity is present in early fractions, it was impossible to quantitate the mitochondrial topoisomerase u_nknotting activity until after the supernatant from the PEG precipitation was fractionated on phosphocellulose. The overall yield was 9% based on the assumption that there was a constant amount of enzyme activity from crude mitochondria to the phosphocellulose chromatography. Throughout the isolation procedure activities for a variety of marker enzymes were determined as previously described (4). As expected, glucose-6-phosphate dehydrogenase activity decreased to non-detectable levels in the PEG-supernatant. Likewise, sulfite oxidase and amine oxidase decreased dramatically while succinic dehydrogenase levels were high in the PEG-supernatant which was derived from the inner membranes. An important indicator of the purity of the mitochondrial preparation and the mitochondrial origin of the topoisomerase isolated is the ratio of DNA polymerase gamma (the mitochondrial matrix enzyme responsible for mtDNA replication) to the DNA polymerase alpha (the nuclear polymerase responsible for chromosomal DNA replication). 692
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I SUMMARY OF THE PURIFICATION OF DNA TOPOISOMERASE II FROM CALF THYMUS MITOCHONDRIA STEP
FRACTION
VOLUME TOTAL SPECIFIC PURIF. YIELD PROTEIN ACTIVITY ACTIVITY (mL) (mg) (UNITS) (U/rag) (FOLD) (%)
I CRUDE MITOCHONDRIA 210
1316
11500
8.7
1
100
965
11500
11.9
1
100
40
197.5
11500
58.2
4.9
100
120
87.7
11500
131
11
100
11.5
0.805 11500 14286
1200
100
VI HYDROXYLAPATITE COLUMN FRACTION
0.66
0.085 1320 15529
1305
11.5
VII DIALYZED FRACTION
0.26
0.028 1040 37142
3121
9.0
II PURE MITOCHONDRIA 1II MITOPLAST IV DILUTED PEG-SUPE V PHOSPHOCELLULOSE COLUMN COMBINED
95
Starting material was 945 grams of fresh calf thymus gland. Preparation of various intermediate fractions and column chromatograaphy was as described in MATERIALS AND METHODS.
A ratio of at least 30:1 gamma:alpha polymerase has been reported in the past as a marker for mitochondrial enzymes (4,16,17). Because D N A polymerases g a m m a and beta
are somewhat active under alpha
polymerase assay conditions, we used aphidicolin, a selective inhibitor of alpha polymerase (14), to determine the amount of contributing beta and gamma activity in our assay. As seen in Table II, the ratio of g a m m a to alpha polymerase increases in each purification step and in the PEG-supernatant it is almost 80:1. This is in accord with previous reports (4,16,17) and, together with the other marker enzyme values, establishes the mitochondrial origin of our enzyme.
The mitochondrial topoisomerase unknots D N A in an ATP-dependent reaction. One of the features of eukaryotic type II topoisomerases is their ability to remove supercoils and/or knots from D N A in the presence but not the absence of ATP. Although relaxation of supercoils in circular D N A can be achieved by either a type I or II topoisomerase, the unknotting of phage P4 D N A is catalyzed by type II but not type I topoisomerases (11). Therefore, this latter activity is considered indicative of a type II topoisomerase. Fig. 1 shows the results of treating knotted P4 DNA with the purified mitochondrial enzyme in 693
V o l . 176, N o . 2, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II DNA POLYMERASE
Fraction
ACTIVITIES
IN M I T O C H O N D R I A L
Polymerase Actvity" alpha
Crude mito
FRACTIONS
Ratio
gamma
gamma/alpha
67
287
4.3/1
Mitoplast
4
72
18/l
PEG supe
1.3
101
78/1
a DNA polymerase activity is shown as units determined as described in Materials and Methods.
the p r e s e n c e and a b s e n c e of A T P . Without a d d e d A T P (lane b) the substrate D N A is u n c h a n g e d c o m p a r e d to u n t r e a t e d control (lane a) which a p p e a r s as a diffuse s m e a r u p o n a g a r o s e gel electrophoresis. With the a d d i t i o n of 1 m M A T P , however, the D N A is extensively u n k n o t t e d a n d a p p e a r s as an intense, slower migrating band (lanes c-f). As the m i t o c h o n d r i a l e n z y m e is diluted this unknotting activity c o r r e s p o n d i n g l y decreases.
abcdef
M-
abc
defg
hi
j k I rn
L-
(D
®
Fig. 1. P4 unknotting assay of mitochondrial DNA topoisomerase II in the presence or absence of ATP. Lane a, P4 DNA only; lane b, plus enzyme minus ATP; lane c-f contain serial diluted enzyme plus 1 mM ATP; lane c, 1 uL; lane d, 1/2 uL; lane e, 1/4 uL; lane f, 1/8 uL. DNA bands labelled M and L are monomer and linear P4 DNA, respectively. Fig. 2. P4 unknotting assay of mitochondriat topoisomerase II in the presence or absence of antitumor drugs. Lane a, P4 DNA only; lane b, P4 DNA plus enzyme plus 1 mM ATP; lane c, same as lane b minus ATP. Lane d-g, same as lane b plus the indicated amounts of m-AMSA: lane d, 0.5 uM; lane e, 5 uM; lane f, 50 uM; lane g, 150 uM. Lane h, same as lane g minus enzyme. Lane i-l, same as lane b plus the indicated amounts of VM-26; lane i, 0.5 uM; lane j, 5 uM; lane k, 50 uM; lane 1, 150 uM. Lane m, same as lane 1 minus enzyme. 694
V o I . 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Response of the mitochondrial topoisomerase to the antitumor drugs, mAMSA and VM-26.
Recently, it has come to light that the type I and II topoisomerases are the
intracellular targets of a number of antitumor drugs (9). For example, the plant alkaloid, camptothecin, has been shown to selectively inhibit the nuclear type I topoisomerase (8). The anticaneer drugs, mAMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidine) and VM26, have been shown to inhibit the nuclear type II topoisomerase (18,19). Since these initial reports, a large number of studies have addressed the biochemical, biological, and physiological consequences of the drug-induced inhibition of topoisomerases. These many studies have concerned, almost exclusively, effects on the nuclear enzymes. There is little known of the response of mitochondrial topoisomerases to these antitumor drugs. We therefore determined the sensitivity of our purified mitochondrial topoisomerase II to two anti-neoplastic drugs, mAMSA and VM-26. Figure 2 shows the results of topoisomerase II assays in the presence and absence of mAMSA and VM-26. There is significant inhibition by as little as 0.5 uM mAMSA and essentially complete inhibition by 50 uM mAMSA (compare lanes d and f to lane b). Likewise, VM-26 inhibits the mitochondrial enzyme significantly at low concentrations with close to complete inhibition by 50 uM (lanes i and k). Both the intercalating drug (mAMSA) and the non-intercalating drug (VM-26) can inhibit the mitochondrial topoisomerase II with a similar profile although it seems that mAMSA may be a slightly more potent inhibitor. The mitochondrial topoisomerase II is more sensitive to mAMSA and VM-26 than the nuclear enzyme. Considering the many previous drug studies and our own results, it is clear that these anti-neoplastic drugs can inhibit all the cellular topoisomerases, those in the
2.0
!\
5
:\ \ \ \ 7"\
~_ 1.s >
I--O
