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Annals of Clinical & Laboratory Science 31:187-198 (2001)
© 2001 Association of Clinical Scientists

Hoechst 33342-Induced Apoptosis is Associated with Decreased Immunoreactive Topoisomerase I and Topoisomerase I-DNA Complex Formation

Xinbo Zhang and Frederick Kiechle
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan

Address correspondence to Frederick Kiechle, M.D., Ph.D., Department of Clinical Pathology, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, Michigan 48073-6769, USA; tel 248 551 8020; fax 248 551 3694; e-mail fkiechle{at}beaumont.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Hoechst 33342, but not Hoechst 33258, induces apoptosis and inhibits topoisomerase 1 activity in vivo. Topoisomerase I relaxes superhelical DNA through a single strand breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site, forming a transient intermediate called a cleavable complex. The fate of cellular topoisomerase 1 in Hoechst 33342-induced apoptosis is unknown. We analyzed the binding capacity of topoisomerase 1 to 32P-labeled plasmid pCI DNA, the immunoreactive topoisomerase 1 concentration and topoisomerase 1 activity in BC3H-1 myocytes and HL-60 cells treated with Hoechst 33342 and Hoechst 33258 by using covalent transfer of 32P radioactivity from plasmid DNA to topoisomerase 1, Western blotting and topoisomerase 1-mediated plasmid relaxation assay, respectively. Hoechst 33342, but not Hoechst 33258, induced topoisomerase 1 dysfunction in both BC3H-1 myocytes and HL-60 cells measured by (1) a decrease in the topoisomerase 1 to DNA binding capacity or cleavable complex formation; (2) a decrease in intracellular concentration of immunoreactive topoisomerase 1; and (3) an inhibition of nuclear endogenous topoisomerase 1 activity. These results suggest that destruction of immunoreactive topoisomerase 1 and topoisomerase 1-DNA complexes or cleavable complexes results in inhibition of topoisomerase 1 activity, a key step in the Hoechst 33342-induced apoptotic process.

(received 5 December 2000; accepted 14 December 2000)

Keywords: Apoptosis, Hoechst 33342, Hoechst 33258, topoisomerase I, topoisomerase I-DNA complex

Abbreviations: TOP1, topoisomerase I; TOP2, topoisomerase II; H342, Hoechst 33342; H258, Hoechst 33258; FBS, fetal bovine serum; MEME, minimum essential medium Eagle; BCA, bicinchoninic acid; SDS, sodium dodecyl sulfate


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Topoisomerases are ubiquitous nuclear enzymes that modify DNA topology during macromolecular synthesis by introducing transient breaks into the helix [1,2]. Two classes of topoisomerases exist in eukaryotic cells: type I enzymes (TOP1), which cause single-strand DNA breaks, and type II enzymes (TOP2), which cause double-strand DNA breaks [3,4]. Based on studies using etoposide or camptothecin, the two prototypic topoisomerase poisons, TOP1- and TOP2-mediated cell death pathways, are associated with the following steps: (1) the processing of stabilized cleavage complexes into frank DNA strand breaks; (2) sensing DNA damage which activates stress-associated signaling pathways and cell cycle arrest; and (3) activation of a preexisting group of enzyme precursors, typified by the cysteine-dependent aspartate-directed proteases (caspases), which catalyze the relatively orderly biochemical cascade of terminal events leading to apoptosis [5–7].

Apoptosis is a genetically mediated mechanism by which individual cells orchestrate their own deletion in normal and diseased tissues. Apoptosis is a complex process which includes signal transduction [8,9] and the degradation of cellular protein and DNA [10]. Although it appears that the downstream biochemical events in apoptosis are the same, such as phosphatidylserine translocation, DNA fragmentation, and activation of caspases, different inducers of apoptosis act on different primary sites within the cell [11]. Determination of the primary cellular sites of action for an inducer of apoptosis is required to determine the specific apoptotic intracellular mechanism(s) that are utilized to cause cell death.

Bisbenzimides [Hoechst 33342 (H342) or Hoechst 33258 (H258)] are cell-permeable, adenine-thymine binding fluorescent dyes, which are widely used to stain DNA for evaluating the cell cycle, apoptosis, and quantifying viable cells by flow cytometry [12,13]. The only structural difference between the two dyes is that the H258 has a hydroxy group where H342 has an ethoxy group. This structural difference confers greater lipophilic properties and greater cell membrane permeability for H342 [14]. H342 and H258 bind to the minor groove of DNA covering at least six base pairs and a minimum of four AT sequences are required for a tight-DNA interaction [15,16]. Utsuno et al [17] report that at higher concentrations of H258, dye binding to DNA is weaker, has no base pair preference, and may involve intercalation. This second binding model has not been confirmed for H342.

Following binding of these drugs to the minor groove of DNA, transcription, replication [18], or the activity of DNA topoisomerase enzymes may be inhibited [16,19,20]. We have reported that H342, but not H258, induces apoptosis [21] when BC3H-1 myocytes were stained with H342 using the staining method described by Lizard et al [13]. H342, but not H258, induces apoptosis in several cell lines including HL-60 cells in a dose-dependent and time-dependent manner [21–24]. H342 initiates apoptosis in BC3H-1 myocytes by a pathway that is independent of de novo RNA and protein synthesis, p53 expression, and activation of serine protease and interleukin converting enzyme [20]. However, H342-induced apoptosis is associated with mitochondrial dysfunction [20], inhibition of endogenous nuclear TOP1 activity [20–24], and reduction of the formation of normal TATA box binding protein/TATA box promoter complexes [25,26], and intracellular accumulation of transcription factor E2F-1[27], suggesting that H342-induced apoptosis is associated with a novel biochemical pathway. The fate of intracellular TOP1 during H342-induced apoptosis is unknown. The present study demonstrates that H342, but not H258, induces TOP1 dysfunction in BC3H-1 myocytes and HL-60 cells including (1) a decrease in cleavable complex formation; (2) a decrease in the intracellular concentration of immunoreactive TOP1; and (3) inhibition of endogenous nuclear TOP1 activity. These results suggest that the destruction of immunoreactive TOP1 and TOP1-DNA complexes that occurs in H342-treated cells, but not in H258-treated cells, results in the inhibition of TOP1 activity that occurs during the apoptotic process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Materials.  Plasmid pCI was purchased from Promega (Madison, WI). Random primed DNA labeling kit, alkaline phosphatase-linked anti-human IgG CSPD ready-to-use (chemiluminescent substrate), and protease K were from Boehringer Mannheim GmbH (Mannheim, Germany). [{gamma}-32P]-dATP was from NENTM Life Science Products (Boston, MA). Acrylamide/bis solution (30%) was purchased from Bio-Rad (Hercules, CA). Bicinchoninic acid (BCA) protein assay reagent was from Pierce (Rockford, IL). H342 and H258 were from Sigma (St. Louis, MO). RPMI 1640 medium and minimum essential medium Eagle (MEME) were purchased from Mediatech, Inc. (Herndon, VA). Fetal bovine serum (FBS) was from Biocell Laboratories (Rancho Dominguez, CA). Bal-31 nuclease was from New England Biolabs, Inc. (Beverly, MA). Calf thymus TOP1 was from Life Technologies (Gaithersburg, MD). Autoradiography was performed with medical X-ray film (Fuji Medical Systems USA, Stamford, CT).

Cell culture and treatment.  The murine muscle cell line (BC3H-1) was grown in MEME with 10% FBS as previously described [28]. The human promyelocytic leukemia cell line HL-60 was grown in RPMI-1640 medium with 10% FBS. For experiments, cells were plated at a density of 1 x 104 cells/ml and were cultured for 2–4 days with 80–90% confluence (BC3H-1 myocytes ) or with 1 x 105 cells/ml density (HL-60 cells) prior to treatment with H342 and H258. H342 and H258 were dissolved in distilled water at 25 mg/ml and added to MEME with 2% FBS at a final concentration of 26.7 µM for 1, 3, 6, 12, and 24 hr.

Preparation of nuclear extracts.  Untreated cells and cells that were dye-treated cells for different time intervals (described above) were rinsed with ice-cold nuclear buffer [150 mM NaCl, 1 mM KH2PO4, 5 mM MgCl2, 1 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 10 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 10% (v/v) glycerol, pH 6.4], scraped, and centrifuged (750 x g). Cells were resuspended in 0.1 volume of ice-cold nuclear buffer, followed by 0.9 volume of cold nuclear buffer containing 0.3% Triton X-100. The mixture was gently rotated for 10 min at 4°C. The nuclei were spun down (750 x g) and resuspended in Triton X-100-free nuclear buffer. To obtain nuclear extracts, all procedures were carried out in the presence of aprotinin (0.44 trypsin-inhibitory unit/ml), as well as 0.1 mM phenylmethylsulfonyl fluoride. Isolated nuclei were washed once in Triton X-100-free nuclear buffer and resuspended in nuclear buffer containing 0.35 M NaCl (final concentration). The salt extraction was performed for 30 min at 4°C with gentle rotation. The nuclei were spun (14,000 x g) and the supernatant was centrifuged again to remove any insoluble materials. Protein concentrations of the nuclear lysates were determined by BCA protein assay reagent. The supernatant (nuclear extract) was used for the DNA relaxation assays and covalent transfer of 32P radioactivity from DNA to TOP1 [20].

Determination of the catalytic activity of TOP1.  Endogenous TOP1 catalytic activities in the nuclear extracts from untreated cells or cells treated with H342 or H258 for different time intervals were examined by the plasmid relaxation assay. Reaction mixtures (20 µl each) containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 µg/ml acetylated BSA, 1 µg of plasmid pCI, and nuclear extract containing 0.5–1 µg total protein were incubated at 37°C for 10 min. One percent sodium dodecyl sulfate ( SDS) and 200 µg/ml protease K were added to terminate the reaction and incubation continued for 30 min at 60°C. The reaction mixtures were analyzed by electrophoresis in an 0.8% agarose gel at room temperature [20].

Covalent transfer of 32P from DNA to TOP1.  The binding capacity of TOP1 to plasmid pCI was determined by covalent transfer of 32P radioactivity from DNA to TOP1 [29]. A 100 µl reaction mixture containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.5 mM dithiothreitol, 30 µg/ml bovine serum albumin, 50 ng of pCI DNA labeled with [{gamma}-32P] dATP by random primed DNA labeling method, and 28 units of calf thymus TOP1 plus H342 or H258 or nuclear extracts (10 µg total protein) was incubated at 37°C for 10 min. The reactions were terminated by adding NaOH to 0.18 M and EDTA to 2.5 mM. After neutralization of the reaction with a precalibrated amount of Tris-HCl, 9 µl of 0.1 M CaCl2 and 7.5 µl of 20% SDS were added, and the sample was adjusted to 300 µl with water. Five units of Bal-31 nuclease were added, and samples were digested for 1 h at room temperature. The reaction was terminated by extraction with 1 volume of phenol. The phenol phase was saved and back-extracted once with an equal volume of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The protein-oligonucleotide complexes were then precipitated from the phenol phase by adding 10 volumes of ice-cold acetone and placing on ice for 10 min. The pellet was dissolved in SDS sample buffer, analyzed by SDS-PAGE, and visualized by autoradiography.

Identification of TOP1 autoantibody in sera of scleroderma patients.  TOP1 is the primary antigen that is recognized by serum from patients with scleroderma [30]. To determine the presence of TOP1 autoantibody in the sera of patients with scleroderma, 1/20 dilutions of sera from two patients with scleroderma and from two patients without scleroderma were incubated with untreated HL-60 cell nuclear extracts containing 1 µg total protein for 30 min in the buffer of plasmid relaxation assay (total volume 20 µl). After incubation, 1 µg of plasmid pCI was added into each reaction mixture and incubated at 37°C for 10 min. One percent SDS and 200 µg/ml protease K were added to terminate the reaction and incubated for 30 min at 60°C. The reaction mixtures were analyzed by electrophoresis in an 0.8% agarose gel at room temperature [20].

Western blotting.  Nuclear extracts from untreated and treated cells were analyzed for immunoreactive TOP1 using human antisera from scleroderma patients [31]. Samples were electrophoresed on 0.6% SDS-polyacrylamide gels and transferred to nitrocellulose. Filters were blocked with 5% dried milk in phosphate-buffered saline for 1 h at room temperature. A 1:500 dilution of patient sera was added to this solution, and the primary hybridization was conducted at room temperature for 1 hr. After three successive washings in 1xTBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.15% Tween 20), an anti-human IgG-alkaline phosphatase conjugate (1:1000 dilution) in 1xTBST was added for 1 hr at room temperature. After three washes in 1xTBST, chemiluminescence was used to detect the alkaline phosphatase conjugate after exposure to X-ray film [32].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
H342 or H258 and in vitro TOP1/DNA complex formation.  Goldman et al [29] reported that H342 stimulated the in vitro formation of TOP1/DNA complexes or cleavable complexes at a low concentration (1 µM) and progressively inhibited complex formation at higher concentrations. To determine the effect of H342 and H258 on the in vitro formation of TOP1/DNA complexes, the covalent transfer of 32P radioactivity from plasmid pCI DNA to TOP1 method was used [29]. In the experiment, 32P-labeled DNA was reacted with TOP1 to form covalent TOP1/DNA cleavable complexes. The covalent TOP1/DNA complexes were digested with Bal-31 nuclease. Regions where TOP1 was covalently bound to DNA were protected from Bal-31 nuclease digestion. As shown in Fig. 1Go, both H342 and H258 stimulated the phosphate transfer, illustrated by the enhanced labeling of TOP1/DNA complexes. At a high concentration (1,000 µM) of H342 or H258, this phosphate transfer reaction was completely inhibited.



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  		Fig. 1. H342 or H258 and in vitro TOP1/DNA complex formation. Calf thymus TOP1 and 32P-labeled plasmid DNA were incubated with different concentrations of H342 or H258 for 10 min. Covalent TOP1/DNA complexes were processed by Bal-31 nuclease digestion and electrophoresed in a SDS-polyacrylamide gel as described in "Materials and Methods." The reaction mixtures containing various inhibitors are labeled at the top of lanes.

 
H342 or H258 and endogenous TOP1/DNA complex formation.  Is binding capacity of intracellular TOP1 to DNA to form TOP1/DNA cleavable complexes altered in H342- or H258-treated cells? To answer this question, we used the covalent transfer of 32P radioactivity from DNA to TOP1 method to determine the binding capacity of plasmid pCI DNA to endogenous TOP1 located in nuclear lysates from cells that had been treated with H342 or H258. After BC3H-1 myocytes and HL-60 cells were treated with 26.7 µM H342 or H258 for different time intervals, nuclear extracts were prepared from the untreated, H342-treated, or H258-treated cells. In contrast to the in vitro H342 study, the capacity for the formation of covalent TOP1-DNA cleavable complexes was decreased in the nuclear lysates from both BC3H-1 myocytes and HL-60 cells treated with H342 when compared with the control group (Fig. 2A and 2BGo). This decrease in the formation of endogenous TOP1/plasmid DNA complexes in the presence of H342 indicates either intracellular TOP1 degradation or intracellular TOP1 binding to genomic rather than radiolabeled plasmid DNA. Similar to the in vitro data (Fig. 1Go), the capacity for the formation of covalent TOP1-DNA complexes was increased in the nuclear lysates from both BC3H-1 myocytes and HL-60 cells treated with H258 when compared with the untreated control groups (Fig. 2A and 2BGo). The maximum number of TOP1/DNA cleavable complexes formed as represented by the greatest staining intensity varied between the two cell lines: BC3H-1 myocytes, 12 hr; HL-60 cells, 3 hr.



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  		Fig. 2. H342 or H258 and endogenous TOP1/DNA complex formation. 32P-labeled plasmid DNA was incubated for 10 min with nuclear extracts from BC3H-1 myocytes and HL-60 cells treated with 26.7 µM H342 or 26.7 µM H258. Covalent TOP1/DNA complexes were processed by Bal-31 nuclease digestion and electrophoresed in a SDS-polyacrylamide gel as described in "Materials and Methods". The reaction mixtures containing nuclear extracts obtained following different treatment times are labeled at the top of lanes. Upper panel (Fig 2A): results from BC3H-1 myocytes; lower panel (Fig. 2B): results from HL-60 cells.

 
Endogenous TOP1 activity in cells treated with H342 or H258.  To determine whether a decreased binding capacity of intracellular TOP1 to DNA to form cleavable complexes is associated with a loss of intracellular TOP1 activity, the plasmid relaxation assay was used to measure endogenous TOP1 activity in nuclear lysates of BC3H-1 myocytes or HL-60 cells after incubation with H342 or H258. Endogenous TOP1 activity was progressively inhibited in cells treated with H342 for different times (Fig. 3A and 3BGo). No significant changes in TOP1 activity were found in nuclear lysates from H258-treated cells (Fig. 3A and 3BGo).



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  		Fig. 3. Endogenous TOP1 activity in the nuclear extracts from H342- or H258-treated cells. The nuclear extracts were prepared from untreated cells or from 26.7 µM H342- or 26.7 µM H258-treated cells. Nuclear extracts (1 µg total protein) were incubated with 1 µg plasmid pCI to determine TOP1 catalytic activity as described in "Materials and Methods." The reaction mixture was then analyzed by electrophoresis in 0.8% agarose gel. Upper panel (Fig. 3A): results from BC3H-1 myocytes; lower panel (Fig. 3B): results from HL-60 cells.

 
Immunoreactive TOP1 concentration in cells treated with H342 or H258.  To determine whether reduced TOP1 enzymatic activity is associated with a decrease in cellular TOP1 concentration and potential degradation of intracellular TOP1, Western blotting was used. The TOP1 antibody was obtained from the sera of patients with scleroderma. To confirm the presence of TOP1 autoantibodies in the sera of these patients, the plasmid relaxation assay was used to detect TOP1 autoantibodies. TOP1 autoantibodies found in scleroderma patients’ sera are known to inhibit TOP1 activity (33). As demonstrated in Fig. 4Go, TOP1 activities in nuclear lysates of HL-60 cells are inhibited after incubation with sera each from one of two different patients with scleroderma, whereas sera each from one of two different patients without scleroderma have no inhibitory effect. In the control cells, the antisera recognized three major proteins, as reported previously (34), including the 100 kDa band which correlates with the predicted molecular weight of TOP1 (Fig. 5A and 5BGo). Immunoreactive TOP1 concentration in cells treated with H342 was significantly decreased after 6 h (BC3H-1 myocytes) and 12 h (HL-60 cells) (Fig. 5A and 5BGo). In contrast, the immunoreactivity of TOP1 in H258-treated groups remained relatively unchanged (Fig. 5A and 5BGo).



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  		Fig. 4. Detection of TOP1 autoantibodies in sera of patients with scleroderma. The plasmid relaxation assay was used. Before the incubation of the nuclear extracts isolated from untreated HL-60 with 1 µg plasmid pCI, the nuclear extracts was incubated with a 1/20 dilution of sera each from one of two patients with scleroderma or sera each from one of two patients without scleroderma for 30 min in plasmid relaxation assay buffer, then added plasmid DNA into each reaction mixture and further incubated for 10 min. The reaction mixtures were then analyzed by electrophoresis in 0.8% agarose gel. pCI, plasmid pCI alone; lane 1 and lane 2, plasmid pCI + nuclear lysates + sera each from one of two different patients without scleroderma; lane 3 and lane 4, plasmid pCI + nuclear lysates + sera each from one of two different patients with scleroderma.

 


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  		Fig. 5. Immunoblot analysis of H342- and H258-induced change in TOP1 protein concentration. BC3H-1 myocytes and HL-60 cells were treated with 26.7 µM H342 or 26.7 µM H258 for different hours indicated (numbers at the top of each lane). Nuclear extracts were isolated from untreated and treated cells, and fractioned on 0.6% SDS-polyacrylamide gels. Western blots were incubated with human sera from scleroderma patients as described in "Materials and Methods." The resulting bands were visualized by chemiluminescence. Fig. 5A (upper panel). Results from BC3H-1 myocytes. Fig. 5B (lower panel). results from HL-60 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Inhibition of TOP1 activity by compounds which intercalate with duplex DNA, like protoberberines or NB-506, or bind to the TOP1/DNA interface, like camptothecin (7) or H342 (15) leads to apoptosis. H342 binds to six base pairs in AT regions of the minor groove of DNA by hydrogen bonding (15). The apoptotic pathway common to both types of compounds is the initial stabilization of the ternary TOP1/DNA/drug cleavable complex which initiates DNA fragmentation secondary to collison with an active replication fork, resulting in fork arrest and double strand fork breakage. These events trigger the cellular metabolic cascade that terminates in apoptosis.

In this paper, we have investigated the fate of TOP1 after the DNA fragmentation process associated with apoptosis has been initiated by H342. Previous studies have demonstrated DNA fragmentation by agarose gel electrophoresis after H342 treatment for 3 h using BC3H-1 myocytes (21) and 6 h using HL-60 cells (23). No DNA fragmentation was observed after H342 treatment for 1 hr (BC3H-1 myocytes) or 3 h (HL-60 cells). This latent period prior to evidence of apoptosis of approximately 3 hr has been observed with other TOP1 inhibitors [7].

The first step in TOP1 catalyzed activity is the binding of TOP1 to DNA to form the reversible TOP1/DNA complex or cleavable complex. We used the covalent transfer of 32P radioactivity from 32P-labeled plasmid pCI DNA to TOP1 to form the covalent TOP1/plasmid DNA cleavable complex [29]. The in vitro addition of H342 or H258 to calf thymus TOP1 and 32P-labeled plasmid pCI DNA led to similar results. There was increase in cleavable complex formation at low concentrations and inhibition of cleavable complex formation at high concentrations (Fig. 1Go). The increase in cleavable complex formation induced by H342 and H258 is attributable to trapping of reversible TOP1-DNA cleavable complexes at AT-rich regions of DNA [16] and the subsequent reduction at 1,000 µM may reflect reduced access of TOP1 to DNA saturated with Hoechst dye [16].

Next, the fate of TOP1/DNA cleavable complex formation in H342 or H258-treated BC3H-1 myocytes (Fig. 2AGo) or HL-60 cells (Fig. 2BGo) was determined. Endogenous TOP1 from nuclear lysates was incubated with 32P-labeled plasmid pCI DNA. Incubation of both cell lines with H258 resulted in an increase in cleavable complex formation similar to the in vitro study (Fig. 1Go). However, incubation with H342 caused a marked decrease in the formation of endogenous TOP1/plasmid DNA complexes (Fig. 2A and 2BGo). There are at least three potential explanations for this decrease in cleavable complex formation. First, the 50-fold greater capacity of the more lipophilic H342 to associate with cellular DNA compared to H258 [14] may reduce TOP1 access to H342-saturated DNA. Second, a greater percentage of H342 may be bound to genomic DNA, not 32P-labeled plasmid pCI DNA, by protein-genomic DNA crosslinks compared to H258.

Previous studies have demonstrated that H342 but not H258 induces protein-DNA crosslinks and DNA strand breaks in cultured mammalian cells in a dose-dependent manner [16,35]. Third, TOP1 may be degraded following the activation of caspases during the apoptotic process and decrease TOP1/DNA complex formation. Treatment of purified TOP1 with caspase-3 resulted in cleavage at DDVD146 Y and EED170 G, whereas treatment with caspase-6 resulted in cleavage at PEDD123 G and EEED170 G [36]. The cleavage sites of TOP1 by caspase-3 and caspase-6 are located at NH2-terminal domain (Met1-Gly214). The elimination of these regions has no effect on the in vitro or in vivo activity of the enzyme [36,37]. In addition, neither cleavage reaction was complete. Thirty percent or 50% of TOP1 remained intact after the 2-hr incubation with caspase-3 or caspase-6, respectively [37]. However, an irreversible inhibitor of caspase-1 (Ac-Try-Val-Ala-Asp-chloromethylketone) and an inhibitor of trypsin-like serine proteases (N -p-tosyl-L-lysine chloromehtyl ketone) did not prevent H342-induced apoptosis in BC3H-1 cells [20]. Further investigation is required to determine if TOP1 is degraded by caspases or other proteases.

The inhibition of endogenous TOP1 activity in nuclear extracts of H342-treated BC3H-1 myocytes or HL-60 cells as measured by the plasmid unwinding assay (Fig. 3A and 3BGo) temporally parallels the reduction in immunoreactive TOP1 measured by Western blotting (Fig. 5A and 5BGo). Human TOP1 is comprised of four major domains: (a) an NH2-terminal domain (approximately 24 kDa) not required for enzymatic activity, (b) the core domain (approximately 54 kDa), (c) a linker region (approximately 3 kDa), and (d) the COOH-terminal domain (approximately 10 kDa), including the active site tyrosine [37,38]. The highly conserved core domain (Met215-Ala635) contains three subdomains that can bind to DNA and cleave DNA [37]. Proteolytic degradation of TOP1 within the DNA binding portion of the core domain could result in loss of TOP1 enzymatic activity, immunoreactivity as well as cleavable complex formation.

The intracellular concentration of TOP1 significantly decreased during H342-induced apoptosis of HL-60 cells and BC3H-1 myocytes without the production of a discrete cleavage fragment or fragments (Fig. 5A and 5BGo). Previous studies have demonstrated different patterns of TOP1 degradation during apoptosis. For example, some studies demonstrated that intracellular TOP1 decreased during etoposide-induced apoptosis in HL-60 cells [5] and camptothecin-induced apoptosis in KB cells [32] without generating detectable TOP1 fragments. TOP1 was degraded into small fragments during tumor necrosis factor-induced apoptosis in C3HA fibroblasts [39]. In contrast to these two findings, other studies demonstrate that a TOP1 fragment of ~70 kDa was produced after a variety of cells were treated with various apoptotic stimuli including etoposide [39], UV-B irradiation [40], anti-CD95 antibody [39,41], and topotecan [37]. However, in all the studies described above, the endogenous TOP1 enzymatic activity or the DNA binding capacity of intracellular TOP1 was not determined [5,32,34,35,37–41]. Since the activity of 70 kDa TOP1 is indistinguishable from that of the native full-length human TOP1 [37], no reduction in TOP1 enzymatic activity would be predicted when this TOP1 fragment was present.

The data presented in this paper demonstrate that after 6 hr of incubation of BC3H-1 myocytes or after 12 hr of incubation of HL-60 cells with 26.7 µM H342, there is a significant reduction or complete suppression of TOP1 binding to DNA (Fig. 2Go) of endogenous nuclear TOP1 activity (Fig. 3Go) and of the nuclear concentration of immunoreactive TOP1 (Fig. 5Go). This temporal relationship suggests that TOP1 is proteolytically degraded in a catalytically required region (perhaps the core region) and/or elsewhere, reducing enzymatic activity and immunoreactivity.

In summary, the catalytic activity of TOP1 can be divided into four steps: (1) binding of TOP1 to DNA; (2) DNA cleavage of one strand associated with covalent attachment of TOP1 to one of the termini of the nicked DNA; (3) single-strand passage; and (4) ligation of the cleaved DNA strand. Therefore, the binding capacity of TOP1 to DNA is a critical step in the modulation of DNA topology [4].

The pathways employed by H342 to induce apoptosis involve a variety of intracellular metabolic perturbations including phosphatidylserine translocation [23], oligonucleosomal fragmentation of DNA typical of apoptosis [20–24,26], increased intracellular concentration of transcription factor E2F-1 [27], and reduction of normal TATA box binding protein/TATA complexes and increased small molecular weight TATA box binding protein/TATA complexes [20,24,26].

The data presented in this paper suggest that the destruction of immunoreactive TOP1 and TOP1-DNA cleavable complexes results in the inhibition of TOP1 activity, another step in the H342-induced apoptotic pathway. Further investigation is required to determine the mechanism of TOP1 degradation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Ms Patricia Schmidt for typing this manuscript. This project was supported by the William Beaumont Hospital Research Institute.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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