Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered DNA topoisomerase II in multidrug resistance

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.     

Mammalian topoisomerase II activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.     

Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II

The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens. To cahracterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16. To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11–2933, was added to the selections. The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold). These cells are also two- to fourfold resistant to m-AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P-glycoprotein substrate vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced ³H-VP-16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X. Using live cells treated with VP-16, band depeletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addijtion, the topoisomerase li present in nuclear edtracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1)inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3. © 1993 Wiley-Liss, Inc.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.     

Topoisomerase II cleavage in chromatin

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.     

Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity.

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.     

Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.     

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors.

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3–4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage in the of VP-16 was, in part, related to a 2–3-fold decrease in both the amount and activity of topisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide).

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.     

5,6-Dichloro-1-beta-O-ribofuranosylbenzimidazole induces DNA damage by interfering with DNA topoisomerase II

We have examined DNA in cells treated with 5,6-dichloro-1-beta-O-ribofuranosylbenzimidazole (DRB), an adenosine analogue. The results show that DRB induces an partial fragmentation of DNA when the cells are lysed in dilute alkali. Fragmentation of DNA does not occur in control cells, nor in cells pretreated with novobiocin or VP-16/VM-26. The data show that DRB interferes with DNA topoisomerase II. In agreement with this interpretation, crude nuclear extracts of DRB-treated cells result in reduced in vitro KC1/SDS precipitation of covalent protein-DNA complexes. Formation of covalent complexes is typical of topoisomerase-DNA interaction.     

Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo.

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.     

DNA topoisomerase II from mammalian mitochondria is inhibited by the antitumor drugs, m-AMSA and VM-26.

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 microM, significantly at 5.0 microM, and completely at 50 microM. 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.     

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.     

Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays

We have characterized the topoisomerase I and II activities in nuclear extracts from immature embryos of Zea mays and the effect of the treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) and abscisic acid (ABA). These extracts were shown to be essentially devoid of protease and nuclease activities and they were tested for their ability to relax supercoiled DNA, unknotting P4 DNA and catenate circular duplex DNA under catalytic conditions. Unknotting and catenation reactions are strictly magnesium- and ATP-dependent, but not the relaxation of circular supercoiled DNA allowing the detection of both topoisomerase I and II activities. Two cytotoxic drugs, camptothecin, a plant alkaloid that inhibits cukaryotic topoisomerase I, and epipodophyllotoxin VM-26 (teniposide) that inhibits topoisomerase II, have been assayed in our extracts showing similar inhibitory effects on topoisomerase enzymes. Alkaline phosphatase treatment of nuclear extracts abolishes both topoisomerase activities. Nuclear extracts from embryos treated with 2,4-D showed 200% increase on topoisomerase II activity as compared with untreated ones, but only residual activity was detected in ABA-treated embryos. Nuclear extracts from hormone-treated and untreated embryos showed similar topoisomerase I activity with deviations of less than 25%. These differences are discussed in terms of possible post-translational modifications of the enzymes associated with the increase in proliferation activity of calli.