In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone.

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.

Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and 180kD forms of topoisomerase II.

The activity of topoisomerase II and the cellular content of the 170kD and 180kD forms of the enzyme were studied as functions of transformation and growth state by using normal and ras-transformed NIH-3T3 cells. Total topoisomerase II activity, as measured by the unknotting of P4 DNA, was higher in ras-transformed than in normal cells in similar growth states, and was higher in exponentially growing than in plateau cells for both cell lines. Total topoisomerase II levels, as measured by immunoblotting, showed a similar dependence on transformation and growth state. The relative amounts of the 170kD and 180kD forms of the enzyme varied as a function of transformation and growth state. The proportion of 170kD topoisomerase II was higher in ras-transformed than in untransformed cells and depended much less on growth state in the ras-transformed cells. The topoisomerase II activity in extracts of ras-transformed cells was more sensitive to inhibition by teniposide and merbarone, drugs which selectively inhibit the 170kD form of topoisomerase II. The ras-transformed cells were also more sensitive to the cytotoxic effects of these drugs. An increase in the relative cellular content of 170kD topoisomerase II is characteristic of ras-transformed 3T3 cells, and the levels of this form of the enzyme appear to be less dependent on proliferation state than in untransformed cells. The susceptibility of certain tumors to killing by topoisomerase II-directed drugs may be due to a higher proportion of 170kD enzyme as well as a higher level of total topoisomerase II activity.

The new partnership of genomics and chemistry for accelerated drug development.

Genomics and combinatorial chemistry are two methods that are revolutionizing drug discovery efforts. Analysis of gene sequences allows identification of novel proteins which are potential therapeutic targets. Recent advances relate to the rate of gene sequencing and data handling resulting from the enormous flow of new gene sequences. Cathepsin K, a cysteine protease involved in bone resorption, was recently identified from a bone cell cDNA library as a potential target for osteoporosis therapy. New sources of chemical agents will rely on advances in high throughput chemical synthesis and structure-based design.

Human osteoclast cathepsin K is processed intracellularly prior to attachment and bone resorption.

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae.

A topoisomerase II-like protein is part of an inducible DNA-binding protein complex that binds 5' of an immunoglobulin promoter.

We previously identified a B cell-specific protein-DNA complex 5' of an immunoglobulin mu heavy chain promoter. The sequences to which this protein complex bound were required for induction of immunoglobulin mRNA levels with interleukin-5 + antigen. Further studies identified a second sequence 5' of these regulatory sequences that bound to both the nuclear matrix and to a similar interleukin-5 + antigen inducible protein complex. Therefore, we sought to identify the putative regulatory proteins that comprised this DNA-binding complex. In this study, we have used anti-topoisomerase II antibodies to demonstrate that one of the proteins found in the interleukin-5 + antigen inducible complexes is serologically related to topoisomerase II. To our knowledge, this is the first report where a topoisomerase II related protein participates in an inducible mobility shifted protein-DNA complex. These data suggest a model in which the enhanced immunoglobulin gene transcription observed after treatment with interleukin-5 + antigen might be explained by the induction of a protein complex that acts to relieve torsional stress along the gene.

Quantitative adaptation of the bacteriophage P4 DNA unknotting assay for use in the biochemical and pharmacological characterization of topoisomerase II.

The ATP-dependent unknotting of phage P4 DNA is a highly specific assay for type II topoisomerases. Despite the unique specificity of the assay, however, its semiquantitative design has limited its use in studying the biochemical properties of these enzymes. To overcome this problem, we have modified the P4 DNA unknotting assay to provide a sensitive and reproducible method for quantifying topoisomerase II activity. Methods are described for accurate measurement of 10-100 ng of unknotted P4 DNA. Under the assay conditions employed, the initial rate of topoisomerase II activity was linear through 30 min. The quantitative assay has been used to determine biochemical and pharmacological parameters of purified topoisomerase II (p170). No topoisomerase II activity was observed in the absence of ATP; enzymatic activity was optimal between 0.5 and 1.0 mM ATP, but substrate inhibition occurred at concentrations above 1 mM. Eadie-Hofstee analysis with varying ATP concentrations gave an apparent Km for ATP of 0.24 mM and a maximal velocity under these conditions of 7.4 ng P4 DNA unknotted/min/ng topoisomerase II. IC50 values were determined for several topoisomerase inhibitors, including amsacrine, teniposide, and novobiocin. Inhibition by teniposide was found to be uncompetitive versus ATP, with a Ki of 3.7 microM. In contrast, inhibition by novobiocin was competitive versus ATP, indicating that teniposide and novobiocin inhibit topoisomerase II by different mechanisms.

DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2.

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation.

Localization of DNA topoisomerase II in Chinese hamster fibroblasts by confocal and electron microscopy.

The localization of the 170- and the 180-kDa isoforms of the enzyme DNA topoisomerase II in growing Chinese hamster fibroblasts has been studied by confocal immunofluorescence microscopy and immunogold electron microscopy after labeling with affinity-purified isoform-specific polyclonal antibodies. Immunofluorescence and immunogold studies, together with quantitative image analysis, show that the two isoforms are present in the nucleoplasm and in the nucleolus. In the nucleoplasm both isoforms are frequently localized at the periphery of heterochromatin regions. In the nucleolus the immunofluorescence and immunogold signals relative to surface area are higher than in the nucleoplasm; both isoforms are localized predominantly in the fibrillar zones. During mitosis both isoforms remain detectable in the cytoplasm. The differential expression of the two isoforms during the cell cycle, observed in other studies, suggests that they have different functions, and their presence in both the nucleoplasm and the nucleolus suggests that these functions are required in both of these nuclear compartments.

Expression of cathepsin K messenger RNA in giant cells and their precursors in human osteoarthritic synovial tissues.

OBJECTIVE: To investigate the expression of cathepsin K messenger RNA (mRNA) in the giant cells found in human osteoarthritic (OA) synovium and associated reparative connective tissues, and to compare this with mRNA expression of cathepsins B, L, and S, which are cysteine proteases known to be highly expressed by cells of the monocyte/macrophage lineage. METHODS: Sections of human OA synovium were processed for in situ hybridization and probed for cathepsins K, B, L, and S. Serial sections were reacted for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, which are selective markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. RESULTS: At 3 sites of monocyte infiltration/giant cell formation (granulation tissue, the intimal and subintimal synovial layers, and deep stroma extending to the periphery of osteophytic tissue), both TRAP-positive mono- and multinucleated cells and TRAP-negative, NSE-positive mononuclear precursors were identified. Cells containing both enzyme activities were also found, potentially indicating an intermediate stage of differentiation. The TRAP-positive mononuclear/giant cells, and the occasional NSE-positive precursor, expressed an intense signal for cathepsin K mRNA, but did not express cathepsins B, L, and S. In contrast, the deep zone of phagocytic-like cells adjacent to sites of ossification expressed high levels of mRNA for cathepsins L, B, and S as well as cathepsin K mRNA. CONCLUSION: Giant cells that form within OA synovial tissue express high levels of cathepsin K mRNA. It appears that cathepsin K acts principally to digest the bone (and cartilage) fragments sheered from the joint surface during OA. The high TRAP activity and the undetectable expression of the macrophage-associated degradative proteases (cathepsins B, L, and S) by synovial giant cells strengthens the hypothesis that cathepsin K is the primary protease involved in bone degradation. At sites of synovial osteogenesis, a population of phagocytic-like cells expressed TRAP and cathepsins B, L, S, and K, and may represent blood-derived macrophages pushed toward an osteoclast phenotype.

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)

Genomic organization and chromosome localization of the human cathepsin K gene (CTSK).

Human cathepsin K is a recently described cysteine protease with high sequence homology to cathepsins S and L, members of the papain superfamily of cysteine proteases. Cathepsin K is abundantly and selectively expressed in osteoclasts and may perform a specialized role in osteoclast-mediated bone resorption. In the present study, the genomic organization and chromosomal localization of human cathepsin K (HGMW-approved symbol CTSK) were determined. Intron-exon boundaries were identified by PCR on human genomic DNA, and subsequently a P1 genomic clone containing the full-length gene was isolated. Cathepsin K spans approximately 12.1 kb of genomic DNA and is composed of eight exons and seven introns. The genomic organization of cathepsin K is similar to that of cathepsins S and L. The gene was mapped to chromosome 1q21 by fluorescence in situ hybridization. Primer walking on the P1 genomic clone identified 1108 bp of 5' flanking sequence and 459 bp of 3' flanking sequence. Ribonuclease protection assay and 5' RACE indicated a single transcriptional start site 49 bp upstream of the initiator Met codon. Analysis of the 5' flanking region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple potential transcription regulatory sites. The characterization of the cathepsin K gene and its promoter may provide valuable insights not only into its osteoclast-selective expression, but also into the molecular mechanisms responsible for osteoclast activation.

Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes.

Several DNA topoisomerase II (Topo II; EC 5.99.1.3) partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.

Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II.

The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.

Characterization of a subline of P388 leukemia resistant to amsacrine: evidence of altered topoisomerase II function.

Sensitive (P388/S) and amsacrine-resistant (P388/amsacrine) sublines of P388 leukemia were cloned in vitro and tested for differential chemosensitivity against a panel of drugs. P388/amsacrine, resistant both in vivo and in vitro to amsacrine, was cross-resistant to other putative topoisomerase II inhibitors including teniposide, etoposide, bisantrene, and doxorubicin. P388/amsacrine, was however, as sensitive as cloned P388/S to camptothecin, an inhibitor of topoisomerase I. The pattern of cross-resistance suggested that an alteration in topoisomerase II may be involved in the resistance of P388/amsacrine to these drugs. No differences in the uptake of amsacrine were detected between the two sublines. Cross-resistance to vinblastine was evident in P388/amsacrine; however resistance to vinblastine was associated with alterations in uptake or efflux of the drug. The number of protein-concealed single-strand breaks induced in whole cells by amsacrine, teniposide, bisantrene, and camptothecin was measured. Diminished numbers of strand breaks in the resistant subline were consistent with decreases in DNA-protein crosslinks. In the absence of drug treatment, resistant cells sustained approximately one-half as many single-strand breaks and DNA-protein crosslinks as the sensitive cells during preparation of nuclei. As measured by the P4 phage DNA unknotting assay, 0.35 M NaCl nuclear extracts from P388/S contained approximately 2.3-fold more topoisomerase II catalytic activity than did extracts from P388/amsacrine. The amount of protein that immunoreacted with a specific antibody to calf thymus topoisomerase II was also decreased in the resistant cells. These data suggest that alterations in topoisomerase II which lead to differential drug sensitivities are partially responsible for the resistance of P388/amsacrine to a specific group of drugs.

Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.

The collagenolytic activity of cathepsin K is unique among mammalian proteinases.

Type I collagen fibers account for 90% of the organic matrix of bone. The degradation of this collagen is a major event during bone resorption, but its mechanism is unknown. A series of data obtained in biological models strongly suggests that the recently discovered cysteine proteinase cathepsin K plays a key role in bone resorption. Little is known, however, about the actual action of cathepsin K on type I collagen. Here, we show that the activity of cathepsin K alone is sufficient to dissolve completely insoluble collagen of adult human cortical bone. We found that the collagenolytic activity of cathepsin K is directed both outside the helical region of the molecule, i.e. the typical activity of cysteine proteinases, and at various sites inside the helical region, hitherto believed to resist all mammalian proteinases but the collagenases of the matrix metalloproteinase family and the neutrophil elastase. This property of cathepsin K is unique among mammalian proteinases and is reminiscent of bacterial collagenases. It is likely to be responsible for the key role of cathepsin K in bone resorption.

Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme.

Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with apparent molecular masses of 180 (p180) and 170 kDa (p170); both proteins unknotted P4 DNA in an ATP-dependent manner and displayed amsacrine-stimulated covalent attachment to DNA. Staphylococcus V8 protease cleavage patterns of p170 and p180 showed distinct differences. Specific polyclonal antibodies to either p170 or p180 recognized very selectively the form of the enzyme used to generate the antibodies. Immunoblotting with these specific antibodies showed that both p180 and p170 were present in cells lysed immediately in boiling sodium dodecyl sulfate. Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amsacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types. The data strongly suggest that p170 is not a proteolytic fragment of p180. Thus, P388 cells appear to contain two distinct forms of topoisomerase II.

Evidence for the participation of topoisomerases I and II in cadmium-induced metallothionein expression in Chinese hamster ovary cells.

CHO-Cdr20 cells are 10-20 times more resistant to killing by cadmium than the parental CHO cells. Resistance has been linked to amplification of the metallothionein genes MT-I and MT-II and their coordinate induction by cadmium and other toxic metals. We studied the roles of the nuclear enzymes topoisomerase I and topoisomerase II in Cd-induced expression of MT-II. Camptothecin-induced DNA strand breakage, mediated by topoisomerase I in cells, increased by approximately 20% when the resistant cells were incubated first with 50 microM Cd and then with camptothecin. Short DNA fragments were enriched in MT-II-hybridizing sequences, indicating that topoisomerase I-associated breakage was directed in part toward the location of induced gene activity. Ten microM camptothecin inhibited Cd-induced accumulation of MT-II mRNA as well as induced and uninduced RNA synthesis in the resistant cells. These data are consistent with the notion that topoisomerase I participates in most or all forms of RNA synthesis. Topoisomerase II inhibitors which trap cleavable complexes (amsacrine, VM-26, VP-16) increased DNA strand breakage at very high concentrations (50-100 microM); the increased breakage appeared to be concentrated near the MT-II gene. This class of inhibitor did not block the accumulation of MT-II message. Novobiocin, a second type of topoisomerase II inhibitor blocked transcription at 300 microM. Merbarone, a novel, third type of topoisomerase II inhibitor, blocked MT-II transcription at 50-100 microM. The latter two inhibited total RNA synthesis in induced, but not uninduced cells. Thus, it is possible that topoisomerase II plays more than one role in transcription and that more than one form of this enzyme is involved.