Introduction

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DNA topoisomerase II (topoII) is an essential nuclear enzyme that alters DNA topology during DNA replication, transcription, recombination, and sister chromatid segregation by the cleavage and religation of double-stranded DNA (Champoux, 1990). Two topoII isoforms exist in mammalian cells [topoII (170 kDa) and topoII (180 kDa)] that are encoded by different genes (Tsai-Pflugfelder et al., 1988; Jenkins et al., 1992). The  isoform plays a role in DNA replication, mitosis, and cell proliferation (Drake et al., 1989; Adachi et al., 1991) whereas the  isoform has been shown to have a critical role in neural development and serves as an important target for the cytotoxic effects of acridines and mitoxantrone (Errington et al., 1999; Yang et al., 2000). DNA topoII serves as a target for many anticancer agents, such as the epipodophyllotoxins (VP-16, VM-26), the anthracyclines (daunorubicin, doxorubicin), and the bisdioxopiperazines (ICRF-187 and ICRF-193) (Osheroff et al., 1994; Sehested and Jensen, 1996). The epipodophyllotoxins and the anthracyclines stabilize cleaved DNA-topoII complexes (Chen et al., 1984), whereas the second class of topoII inhibitors, the bisdioxopiperazines, serve as catalytic inhibitors of topoII by locking topoII on the DNA, thereby preventing its cleavage (Andoh, 1998). Recently, it was suggested that this closed-clamp form of topoII induces cell death through a novel mechanism involving impediment of DNA metabolic events (Jensen et al., 2000). Both classes of topoII inhibitors were also recently found to induce SUMO-1 conjugation (Mao et al., 2000). Because the sensitivity of human tumor cells to these topoII-targeting agents correlates with alterations in topoII expression levels (Beck et al., 1994, 1999; Nitiss and Beck, 1996), understanding the mechanisms that regulate topoII gene expression may ultimately lead to novel strategies for overcoming antitopoisomerase drug resistance.

TopoII gene expression levels are regulated through the cell cycle and can be affected by external stimuli such as heat shock and drug treatments or by cell density-induced growth arrest. For example, in terms of cell cycle, topoII levels accumulate during the S and G₂/M phases, with maximal levels at mitosis, and decrease during the G₀/G₁ phase (Heck et al., 1988; Woessner et al., 1991). Cell cycle-dependent topoII transcription is believed to be mediated in part by cis-acting elements in the topoII promoter that either activate or repress topoII transcription. One study revealed that an inverted CCAAT element (ICE) (nt 108 to 104) has a stimulatory role in topoII promoter activity in proliferating cells (Isaacs et al., 1996), characterized by increased binding of a proliferation-induced protein complex to this ICE. In contrast, another ICE (nt 68 to 64) was found to have a role in topoII transcriptional repression (Falck et al., 1999), because enhanced protein binding to this element correlated with topoII down-regulation in serum-starved cells. This same ICE has also been associated with both p53-mediated transcriptional repression (Wang et al., 1997b) and heat shock-mediated transcriptional activation (Furukawa et al., 1998) of the topoII promoter. The transcription factor c-MYB was also found to trans-activate the topoII promoter and to play an essential role in the regulation of topoII expression during maturation of hematopoietic cell lines (Fraser et al., 1995; Brandt et al., 1997).

In contrast to these observations, however, the mechanisms by which topoII expression levels are altered during the development of drug resistance have not been studied as thoroughly. Several factors that can directly affect gene expression include mutations, altered methylation patterns in gene promoters, and altered regulation of trans-acting factors. Studies from one laboratory implicated the overexpression of the Sp3 transcriptional repressor in the down-regulation of topoII mRNA levels of an etoposide-resistant KB cell line (Kubo et al., 1995; Takano et al., 1999), whereas work from this laboratory reported that reduced expression of Sp3 contributed to decreased topoII expression in a merbarone-resistant CEM cell line (Mo et al., 1997). Moreover, decreased activity of the transcription factor, CP-1 (NF-Y) resulted in transcriptional down-regulation of topoII in a cell line selected for resistance to doxorubicin (Wang et al., 1997a).

To better understand the mechanisms involved in the regulation of topoII gene expression, we examined topoII expression levels, topoII promoter activity, and nuclear protein binding to the topoII promoter in our leukemic CEM cell line selected for resistance to ICRF-187, CEM/ICRF-8. The CEM/ICRF-8 cell line expresses increased topoII protein at the transcriptional level compared with CEM (Morgan et al., 2000). Analysis of the topoII promoter elements in these ICRF-187-sensitive and -resistant cells, described herein, revealed that the third inverted CCAAT element (ICE3) has a key regulatory role in the basal transcriptional activity of topoII that is differentially recognized by a member of the family of NF-Y transcription factors in the parental CEM compared with our ICRF-187-resistant CEM cell line.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Lines. Human leukemic CEM cells and the ICRF-187-resistant subline CEM/ICRF-8 were cultured as described (Morgan et al., 2000). All cell lines were incubated at 37°C in a humidified chamber containing 5% CO₂/95% air.

Western Blot Analysis. Nuclear extracts were prepared from logarithmically growing cells (5 × 10⁵ cells/ml) as described previously (Mo and Beck, 1997). For analysis of NF-YA, NF-YB, and YB-1 transcription factors, proteins (100 µg/well) were separated on a 10% SDS-polyacrylamide gel, electrophoretically transferred onto nitrocellulose, and incubated with either purified mouse anti-NF-YA monoclonal antibody (PharMingen, SanDiego, CA), rabbit anti-NF-YB polyclonal antibody (Biodesign International, Kennebunk, ME), or rabbit anti-YB-1 antibody (generously provided by Dr. Manfred Dietel, Humbolt University, Berlin, Germany). Bound antibody was detected using the enhanced chemiluminescence (ECL) detection method (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer's instructions. Autoradiographic signals were quantified by densitometric scanning using a GS-700 Imaging Densitometer and Molecular Analyst Software (Bio-Rad, Hercules, CA). Equal loading of total cellular protein was determined by stripping and reblotting the membrane with anti--actin antibody (Oncogene Science, Cambridge, MA).

Construction of TopoII Promoter-Luciferase Plasmids for Transient Expression. The recombinant plasmid p557 that contains the full-length topoII promoter (nt 557 to +90) and recombinant plasmids containing serial deletions of the full-length promoter: p382 (nt 382 to +90), p252 (nt 252 to +90), p182 (nt 182 to +90), p162 (nt 162 to +90), and p90 (nt 90 to +90) were provided by Dr. Q. Wang (Fibrogen, Inc., San Francisco, CA; Wang et al., 1997b). Bases are numbered with respect to the major transcription start site (designated +1) (Hochhauser et al., 1992).

Transient Transfections and Luciferase Reporter Assays. DNA was introduced into the cells by electroporation using the Gene Pulser II apparatus with an extender (Bio-Rad), according to the manufacturer's instructions. Each luciferase plasmid was cotransfected with pSV--galactosidase control vector (Promega) into CEM and CEM/ICRF-8 cells and cell extracts were prepared after electroporation as described previously (Morgan et al., 2000). Luciferase activity was measured by a luminometer with an auto-injector (Model TD-20/20; Turner Designs, Sunnyvale, CA). Luciferase activities were normalized to -galactosidase activities.

Preparation of Nuclear Extracts. Nuclear extracts were prepared from logarithmically growing cells (5 × 10⁵ cells/ml). Cells were resuspended in ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 6 mM MgCl₂), incubated on ice for 10 min and centrifuged (1,200g, 5 min at 4°C). The supernatant was removed and the pellet was incubated in buffer A containing 0.5% Nonidet P-40. After 5 min on ice, the nuclei were sedimented (3,300g, 15 min at 4°C) and the nuclear pellets were lysed by the addition of ice-cold buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2.5 µg/ml pepstatin, 10 µg/ml trypsin inhibitor, and 1 mM benzamidine). The nuclear extracts were incubated on ice for 30 min with occasional vortexing, centrifuged (15,000g, 15 min at 4°C), and the cleared supernatant was transferred to a new tube.

Electrophoretic Mobility Shift Assays (EMSAs). Nuclear extracts (15 µg of protein/assay) were incubated for 30 min at room temperature in a 20-µl reaction mixture containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM EDTA, 8% glycerol, 1 µg poly(dI-dC), 20 µg bovine serum albumin, and ³²P-labeled double-stranded oligonucleotide probe (0.2 pmol). The oligonucleotides were labeled with ³²P using Klenow polymerase. Competition for protein binding activity was carried out in the presence of a 50-fold molar excess of unlabeled wild-type or mutant oligonucleotide probe. DNA-protein complexes and free DNA were applied to 4% nondenaturing polyacrylamide gels and separated by electrophoresis at 100 V for 2.5 h in a buffer containing 25 mM Tris, 190 mM glycine, and 1 mM EDTA. The gels were dried and exposed to X-ray film with intensifying screens. For supershift assays, approximately 2 µg of a rabbit anti-NF-YB antibody (Biodesign International) were preincubated with nuclear extract for 1 h at room temperature before the addition of ³²P-labeled oligonucleotide probe. The following DNA oligonucleotides, along with their complementary strands were synthesized (Life Technologies, Gaithersburg, MD) and used for EMSA analysis: ICE3 (5'-CCTCCCTAACCTGATTGGTTTATTCAAACAAACC-3', nt 189 to 156 of topoII promoter); mutant ICE3 (5'-CCTCCCTAACCTGAgTaGTTTATTCA AACA AACC-3', nt 189 to 156); ICE4 (5'-GGTGAGCCCTTCTCATTGG CCAGATTCCC-3', nt 273 to-245); and ICE5 (5'- GGGATCTTAAATAGATTGGCAGTTCCTGGAG-3', nt 407 to 377). The criteria for assigning bands as nonspecific versus specific ICE-protein complexes is based on two controls. First, a DNA-protein complex is specific if it is eliminated by the addition of a molar excess of unlabeled competitor DNA of the same sequence but not by the addition of a molar excess of the same sequence containing mutation(s). Second, the specificity of the band shift is further confirmed when no DNA-complex is formed when the mutated ³²P-labeled oligonucleotide alone is used in the EMSA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

TopoII Promoter Activity Is Increased in the ICRF-187-Resistant CEM/ICRF-8 Cells. We have recently demonstrated that the levels of topoII protein in the CEM/ICRF-8 cells are increased ~ 5-fold compared with the parental CEM cells (Morgan et al., 2000). Similarly, we have also demonstrated by Northern blot analysis that CEM/ICRF-8 cells express greater than 2-fold increase in topoII mRNA (Morgan et al., 2000). To determine whether the increased expression levels of topoII are caused by transcriptional up-regulation, basal transcriptional activity of the full-length topoII promoter was measured by transiently transfecting into drug-sensitive and -resistant cells a construct containing the wild-type topoII promoter (p557) fused to the luciferase reporter gene. These experiments revealed that CEM/ICRF-8 expressed approximately 2.3-fold more promoter activity than the parental CEM cells (Table 1, plasmid p557). These results are consistent with our previous data (Morgan et al., 2000) and suggest that the up-regulation of topoII in the drug-resistant cells occurs in part at the transcriptional level. Importantly, because sequence analysis of the full-length topoII promoter from CEM/ICRF-8 compared with CEM revealed no mutations (data not shown), we suggest that the observed transcriptional up-regulation of the topoII gene in the drug-resistant cells may be caused by alterations in trans-acting factors of the transcriptional machinery.

View this table: [in this window] [in a new window]   TABLE 1 Percentage of luciferase activity of topoII promoter deletion constructs in CEM and CEM/ICRF-8 cells. All data are derived from Fig. 3B. See legend to Fig. 3 for details.

Determination of the Promoter Sequences Responsible for Mediating TopoII Up-Regulation in ICRF-187-Resistant Cells. To identify the promoter sequences responsible for enhanced topoII transcriptional activity in the drug-resistant cells, we measured basal luciferase activity in CEM and CEM/ICRF-8 cells transiently transfected with a series of topoII promoter-luciferase deletion constructs (Fig. 1). Sequence analysis of the topoII promoter has revealed the presence of five ICEs (Hochhauser et al., 1992), numbered 1 through 5. The CCAAT sequence is a common cis-regulatory element; mutation of this motif has been shown to alter the transcriptional activity of eukaryotic genes (Santoro et al., 1988; Hochhauser et al., 1992). Stepwise deletion of the 5' topoII promoter sequences, which includes stepwise deletion of each ICE (Fig. 1), resulted in a concomitant decrease in luciferase activity in both the CEM and CEM/ICRF-8 cells (Table 1). This overall pattern of basal promoter activity in Table 1 is consistent with previous reports (Hochhauser et al., 1992; Wang et al., 1997b) and confirms that ICEs play an important role in the transcriptional regulation of topoII expression.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Schematic map of the human topoII promoter-luciferase reporter constructs. Plasmids containing serial deletions from the 5' end of the human topoII promoter were provided by Dr. Q. Wang (Wang et al., 1997). The sequence numbers depicted correspond to those published by Hochhauser et al. (1992), with the transcription start site set to +1 (indicated by arrow). The positions of the ICEs are indicated by the circles and are numbered (1-5) beginning with the ICE nearest to the +1 transcription start site. The full-length topoII promoter is represented by plasmid p557.

An Inverted CCAAT Element in the TopoII Promoter Plays a Role in the Down-Regulation of Promoter Activity in CEM and CEM/ICRF-8 Cells. To better determine the roles of ICE3, ICE4, and ICE5 in the regulation of topoII expression, CEM and CEM/ICRF-8 cells were transiently transfected separately. Each of the topoII promoter-luciferase constructs contained a mutated ICE3, ICE4, or ICE5. As shown in Fig. 2, point mutation of ICE3 in the context of the full-length topoII promoter resulted in an increase in topoII transcriptional activity (1.33-fold) in the drug-sensitive CEM cells. Although this increase seems small, it is statistically significant as determined by the Student's t test, suggesting that ICE3 may have an important role in topoII expression by negatively regulating basal promoter activity. In the case of the drug-resistant cells, CEM/ICRF-8 exhibited ~2-fold more wild-type topoII promoter activity compared with CEM. Importantly, this activity did not significantly change when ICE3 was mutated (Fig. 2). From these data, we conclude that (a) ICE3 may serve as a negative cis-acting element, and (b) because enhanced transcriptional activity in CEM/ICRF-8 is independent of a wild-type ICE3 sequence, then there may exist alterations in transcription factor binding to ICE3 in the drug-resistant cells.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   ICE3 negatively regulates topoII promoter activity in CEM cells. CEM and CEM/ICRF-8 cells were transiently transfected with a wild-type, full-length topoII promoter-luciferase plasmid (p557) or with the same plasmid containing a mutated ICE3 (p557-mt ICE3), along with the pSV--galactosidase plasmid. Luciferase activity was assayed and normalized to -gal activity 17 h after electroporation as described under Materials and Methods. Data are expressed as a percentage of the corrected luciferase activity of CEM cells transfected with p557, and are averages of three independent experiments (bars, SE). P < 0.05 for p557-mt ICE3 compared with p557 in CEM cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of mutations in ICE4 or ICE5 in the topoII gene promoter on transcriptional activity. A, CEM and CEM/ICRF-8 cells were transiently transfected with either the wild type luciferase reporter plasmid (p382) or with p382 containing a mutation in ICE4 (p382-mt ICE4), along with the pSV--galactosidase plasmid. B, CEM and CEM/ICRF-8 cells were transiently transfected with either the wild type luciferase reporter plasmid (p557) or the p557 containing a mutation in ICE5 (p557-mt ICE5), along with the pSV--galactosidase plasmid. In both A and B, luciferase activity was assayed and normalized to -gal activity 17 h after electroporation as described under the Materials and Methods. Data are expressed as a percentage of the corrected luciferase activity of CEM cells transfected with wild type plasmid, and are averages of three independent experiments (bars, SE).

Differential Binding of Protein Complexes to ICE3 in the TopoII Promoter from CEM and CEM/ICRF-8 Cell Lines. To further explore the role of ICE3 in the regulation of topoII gene expression, protein factor binding to this element was analyzed in CEM and CEM/ICRF-8 by EMSA using a radiolabeled double-stranded DNA oligonucleotide spanning ICE3 (nt 187 to 155). Nuclear extracts derived from CEM and CEM/ICRF-8 cells contained a factor that specifically bound to the ICE3 probe (Fig. 4, lanes 1 and 6). This DNA-protein complex was eliminated by the addition of 50-fold molar excess of unlabeled competitor DNA of identical sequence (Fig. 4, lanes 2 and 7) but not by the addition of 50-fold molar excess mutated ICE3 probe (Fig. 4, lanes 3 and 8). No DNA-protein complex was formed when the mutated ICE3 oligonucleotide alone was used in the EMSA (Fig. 4, lanes 4 and 9), confirming the specificity of this band shift. The addition of an antibody against NF-YB, a specific ICE-binding factor (Hooft van Huijsduijnen et al., 1990; Maity et al., 1992), resulted in both inhibition of complex formation and a supershift (Fig. 4, lanes 5 and 10). At a longer gel exposure time, the antibody supershift became more apparent in extracts derived from CEM/ICRF-8 (data not shown). The major nonspecific band seems to decrease in intensity with NF-YB antibody coincubation. Given that we are using a polyclonal antibody, it is possible that the anti-NFYB antibody cross-reacts nonspecifically with other protein in the prepared cell extracts, thus resulting in the inhibition of nonspecific DNA-protein complex formation. As a control, the addition of a nonspecific rabbit polyclonal antibody of the same isotype as anti-NF-YB gave no supershift (data not shown). These results suggest that NF-YB makes up at least one component of the ICE3 protein-binding complex. Our gel shift profiles showing ICE-protein complexes and NF-YB supershifts are in agreement with others (Isaacs et al., 1996; Herzog and Zwelling, 1997; Furukawa et al., 1998). Interestingly, although the result is qualitatively similar to that obtained using extracts from the parental CEM cells, the level of DNA-protein complexes formed was lower in the ICRF-187-resistant cell line (Fig. 4, compare lane 1 to lane 6). These quantitative differences are not caused by differences in cell growth in culture or in extract preparation because only cells growing logarithmically were used for each independent experiment, and all have yielded the same results.

View larger version (85K): [in this window] [in a new window]   Fig. 4.   Electrophoretic mobility shift assay using nuclear extracts derived from CEM and CEM/ICRF-8 cells. Nuclear extracts (15 µg) derived from CEM or CEM/ICRF-8 cells were incubated (30 min, 25°C) with a 32P-labeled oligonucleotide that unless otherwise stated contained the wild-type ICE3 probe. The DNA-protein complexes and free DNA probe were separated on a 4% nondenaturing polyacrylamide gel and radioactivity was detected as described under Materials and Methods. Lanes 1 to 5 contained extract from CEM cells, whereas lanes 6 to 10 contained extract from CEM/ICRF-8 cells. Lanes 1 and 6, extract alone; lanes 2 and 7, extracts incubated with 50-fold molar excess of unlabeled ICE3; lanes 3 and 8, extracts incubated with 50-fold molar excess of unlabeled mutant ICE3; lanes 4 and 9, extracts incubated with the mutant ICE3 probe only; lanes 5 and 10, extracts incubated with an antibody (2 µg) against NF-YB. The positions of the free probe, nonspecific DNA-protein interactions, ICE3-protein complex, and antibody supershifts are shown on the right. See Materials and Methods for details. This EMSA is representative of three independent experiments.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Electrophoretic mobility shift assay using increasing molar amounts of ICE3 probe. A, nuclear extracts (15 µg) derived from CEM or CEM/ICRF-8 cells were incubated with 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, or 1.4 pmol of a 32P-labeled ICE3 oligonucleotide (lanes 1-7 for CEM cells or lanes 8 to 14 for CEM/ICRF-8 cells, respectively). The DNA-protein complexes and free DNA probe were separated on a 4% nondenaturing polyacrylamide gel, and the gel was dried and exposed to X-ray film. The positions of the free probe, nonspecific DNA-protein interactions, and the ICE3-protein complexes are shown on the left. Shown is a representative of three independent experiments. See Materials and Methods for details. B, shown is the quantification by densitometric scanning of ICE3-protein complexes formed as derived from Fig. 5A.

Analysis of Protein Expression Levels of the CCAAT Box Binding Factors NF-YA, NF-YB, and YB-1 in CEM and CEM/ICRF-8 Cells. By Western blot analysis, the protein levels of the CCAAT box binding factors, NF-YB and NF-YA (Li et al., 1992; Mantovani et al., 1992), were similarily expressed in CEM and CEM/ICRF-8 cells (Fig. 6). The apparent sizes of the isoforms for NF-YB (~32 and 34 kDa) and for NF-YA (~42 and 44 kDa) in Fig. 6 are in agreement with previously published results (Li et al., 1992; Mantovani et al., 1992). Another known CCAAT binding factor includes a 35-kDa YB-1 protein (Didier et al., 1988), which was also present in equal amounts in the CEM and CEM/ICRF-8 cells (Fig. 6). Equal loading of nuclear protein was confirmed by blotting for -actin (data not shown). Thus, our observations suggest that the transcriptional up-regulation of topoII in the drug-resistant cells is not caused by altered expression of these trans-acting factors, suggesting that such alterations as mutations in or post-translational modifications of NF-Y or other binding proteins in the complex may exist.

View larger version (60K): [in this window] [in a new window]   Fig. 6.   Expression of CCAAT box binding factors in CEM and CEM/ICRF-8 cells. Nuclear extracts derived from CEM and CEM/ICRF-8 cells were electrophoresed in a 10% SDS-polyacrylamide gel. NF-YA, NF-YB, and YB-1 were detected by immunoblot using antibodies as described in Materials and Methods. Equal loading of nuclear protein was detected by staining the transferred membrane with Ponceau Red or by blotting for -actin. Each panel is representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have shown recently that topoII expression is up-regulated at both the protein and mRNA levels in a leukemic CEM cell line (CEM/ICRF-8) selected for resistance to the topoII catalytic inhibitor, ICRF-187 (Morgan et al., 2000). Here, we demonstrate that this up-regulation of topoII occurs at the transcriptional level because topoII promoter activity was significantly increased in CEM/ICRF-8 compared with CEM. We have localized the region of the promoter (nt 557 to 162) that seems to be responsible for topoII transcriptional up-regulation in the drug-resistant cells. An ICE located between nt 182 and 162 (ICE3) may play a role as a negative cis-acting element in our leukemic cell lines. We base this conclusion on the following: 1) Using EMSA, ICE3 was recognized by a NF-YB-containing protein complex and this DNA-protein complex was present at a reduced level in the topoII-overexpressing ICRF-187-resistant cells compared with the drug-sensitive cells, and (2) point mutation of ICE3 resulted in a significant increase in topoII transcriptional activity in the drug-sensitive CEM cells.

ICEs are known to be involved in either up- or down-regulating topoII gene expression. For example, wild-type p53 represses topoII gene expression through functional interaction with ICE1 (Wang et al., 1997b). Moreover, ICE1 was involved in topoII transcriptional repression in serum-starved cells (Falck et al., 1999), and heat-shock activation of topoII required a decrease in nuclear binding activity to ICE1 (Furukawa et al., 1998). In contrast, ICE2 was identified as a positive regulatory element for topoII expression in proliferating cells (Isaacs et al., 1996).

Results here suggest that the up-regulation of topoII transcriptional activity in the ICR F-187-resistant cells may be caused in part by differential binding of the NF-Y family of transcription factors to a region of the topoII promoter that contains the third inverted CCAAT cis-acting element. The presence of ICE3 binding activity has been reported (Herzog and Zwelling, 1997; Wang et al., 1997a), but ours is the first study to demonstrate differential ICE3 binding activity as a function of drug-resistance. Accordingly, we offer the suggestion that transcription factor binding to ICE3 negatively regulates topoII expression in CEM cells, possibly by displacing positive regulatory factors flanking this region of the topoII promoter. In this regard, one study has shown that NF-Y DNA binding activity seems to induce bends in the DNA and the extent of bending depends upon the promoter sequences flanking the CCAAT box (Ronchi et al., 1995). The putative negative role of an ICE is further supported by the observation made by Takano et al. (1999) that binding of a nuclear protein factor to ICE1 down-regulates topoII gene expression in an etoposide-resistant cell line.

In terms of our ICRF-187-resistant cells, regulation of topoII expression seems to be attenuated by dissociation of these NF-Y-containing nuclear proteins from the ICE3 promoter region. Altered binding is not caused by mutations in the topoII promoter, because sequence analysis revealed a wild-type promoter in the ICRF-187-resistant cells, just as in the drug-sensitive CEM cells. Furthermore, by Western blot analysis, there were no apparent differences in the levels of the CCAAT box binding proteins, NF-YB, NF-YA, or YB-1 in drug-sensitive and -resistant cells that could account for changes seen in nuclear protein-ICE3 complex formation. These observations suggest that in CEM/ICRF-8, such alterations as mutations in or post-translational modifications of NF-Y or other binding proteins in the complex may exist. If this were the case, then protein-protein interactions and protein-DNA binding activity may all be affected. In the case of interactions between NF-Y and histones, a recent study has suggested a model in which NF-Y interacts/recruits histone acetyltransferases to the promoter, thereby stimulating histone acetylation and activating G₂/M-dependent transcription of the topoII gene (Adachi et al., 2000). Studies are currently underway to determine whether differential phosphorylation and/or gene mutation in CCAAT box binding proteins, and differential histone acetylation, would alter transcription factor binding to the topoII promoter in our ICRF-187-resistant cells.

Although our data presented herein suggest that ICE3 is a negative regulator of topoII expression, it does not seem to be the sole contributor to the altered regulation of topoII expression in the ICRF-187-resistant cells, as suggested by our mutational analysis in Fig. 2. The topoII promoter fragment (nt 557 to 162) found responsible for topoII transcriptional up-regulation in the drug-resistant cells contains ICE3, ICE4, and ICE5. Although there were no differences in the amount of ICE4 or ICE5 protein-complexes formed between the drug-sensitive and -resistant cells as reported by EMSA (data not shown), the possibility exists that promoter elements further upstream or downstream of these ICEs may cooperate with these cis-acting elements to enhance topoII expression in the drug-resistant cells. Additional changes in chromatin structure and/or methylation status within specific regions of the topoII promoter may also contribute to the altered topoII expression observed here; studies examining some of these possibilities are currently underway.

We have also shown here that ICE1 and ICE2 are neither necessary nor sufficient for topoII up-regulation in CEM/ICRF-8 cells. It is not known what distinguishes one ICE from another, but the specific sequences and regulatory promoter elements flanking each ICE that contribute to the overall regulation of inverted CCAAT boxes may be involved. Indeed, by EMSA analysis, the ICE5 oligonucleotide seems to bind to a different nucleoprotein complex in cell extracts compared with the ICE3 and ICE4 oligonucleotides (Herzog and Zwelling, 1997; S.E.M. and W.T.B., unpublished observations). In addition to ICEs, other cis-acting promoter elements have been known to affect topoII expression (Fraser et al., 1995; Brandt et al., 1997). Larger DNA fragments of ~200 bp each, spanning the entire topoII promoter, were used in EMSAs to determine whether cis-acting promoter elements, independent of ICEs, play a role in topoII up-regulation in CEM/ICRF-8, but we have obtained no clear evidence of differential DNA-protein interactions outside of ICE3 in CEM or CEM/ICRF-8 cells (data not shown).

In the context of drug resistance, very little is known about the factors that regulate topoII expression at the transcriptional level. Our study is the first to show that topoII transcriptional up-regulation in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoII promoter. Furthermore, we have demonstrated that ICE3 seems to play a repressive role in topoII expression. Our present studies involve characterization of additional ICE3-binding proteins and cis-acting elements and delineation of differences in the overall chromatin structure of the topoII promoter that may account for topoII over-expression in our ICRF-187-resistant cells.