c-Myb trans-activates the human DNA topoisomerase IIalpha gene promoter.

DNA topoisomerase IIα (topo IIα) is an essential proliferation-dependent nuclear enzyme which has been exploited as an anti-tumor drug target. Since the proliferative status of human leukemia cells is associated with expression of the c-myb proto-oncogene, c-Myb was investigated as a trans-activator of the topo IIα gene. Using topo IIα promoter-luciferase reporter plasmids, c-myb expression caused trans-activation of the topo IIα promoter a maximum of ∼4.5-fold over basal levels in HL-60 human promyelocytic leukemia cells. Trans-activation was submaximal with higher levels of c-myb expression plasmid but a Myb protein lacking its negative regulatory domain resulted in ∼19-fold trans-activation. Mutagenesis and 5′-deletion studies revealed that Myb trans-activation was mediated via a Myb-binding site at positions −16 to −11 and that this region governed the bulk of basal topo IIα promoter activity in human leukemia cells. Trans-activation of topo IIα by c-Myb was lymphoid- or myeloid-dependent. However, B-Myb, a more widely-expressed Myb family member, caused topo IIα trans-activation in both HL-60 cells and HeLa epithelial cervical carcinoma cells. These data provide evidence for a new Myb-responsive gene which is directly linked to and required for cellular proliferation.

DNA topoisomerase II (topo II) 1 is a nuclear enzyme whose catalytic activity is absolutely required for cellular proliferation. The enzyme catalyzes the relaxation and decatention of DNA as a result of its unique coordination of double-strand DNA breakage, strand passage, and religation activities (reviewed in Refs. 1 and 2) via an ATP-modulated, protein-clamp intermediate (3,4). Roles for topo II catalytic activity have been demonstrated or proposed in DNA replication, transcription, and chromosomal segregation (5)(6)(7). Its role in decatenating newly replicated DNA has been shown to be essential for cellular survival in yeast (5,8,9). In addition, nuclear protein-protein interactions with topo II may have implications for modulating its catalytic activity (10) and in maintaining stability of the genome (11).
Two forms of mammalian topo II have been identified (␣ and ␤), each encoded by distinct genes located on separate chromosomes (12). The ␣ form of the enzyme has been the most intensively studied since it is usually the more abundant form in proliferating cells (13). Not surprisingly, topo II␣ expression has been linked to cellular proliferative status in nearly every system studied (14 -18). However, little is known regarding the transcriptional mechanisms underlying this proliferation-dependent expression of topo II␣. Recent data suggest that the NF-Y transcription factor family may be responsible for loss of topo II␣ expression in confluence-arrested cells (19).
In previous investigations, HL-60 leukemia cells have proven an excellent model in which to study topo II␣ transcriptional regulation during the transition from growth to terminal differentiation (20). In these studies, promoter-reporter deletion analysis of the topo II␣ 5Ј-flanking region revealed the presence of a consensus c-Myb-binding site within the proximal promoter (20). c-Myb has therefore emerged as the first candidate factor for regulating topo II␣ gene expression in proliferating hematopoietic cells.
The viral myb oncogene was described independently in two avian retroviruses: avian myeloblastosis virus and the E26 virus (21). The v-Myb protein was originally shown to be a sequence-specific DNA-binding transcriptional activating protein (22) transduced from a normal cellular progenitor, c-Myb (23). Cellular and viral Myb proteins can bind a variety of sequences but the consensus that has emerged is TAAC(G/T)G or TAACNG (22,24,25). Disruption of DNA binding activity is most commonly accomplished experimentally by mutating the TAAC core (26). Viral Myb proteins were found to be truncated at both the C and N termini, implying that normal regulation of this protein in non-transformed cells occurred via these regions and that transformation was a result of their truncation (27). As might be predicted, the cellular form of Myb is also implicated in normal cellular proliferation, albeit in a more regulated fashion. Specifically, the expression of c-Myb is closely linked to proliferative status in normal hematopoiesis; c-Myb is down-regulated following terminal differentiation to monocytes, neutrophils, or erythrocytes (28,29). Antisense oligonucleotides to c-myb inhibit myeloid cell proliferation (30) while overexpression of c-Myb protein is known to inhibit cellular differentiation in erythroid and myeloid cell lines (31,32). In vivo, transgenic mice homozygous for a null c-myb allele do not survive past gestation day 15 due to their lack of hepatic hematopoiesis (33). More directed, tissue-specific inactivation of c-Myb in transgenic mice expressing dominant negative c-Myb peptides in immature T cells blocked thymopoiesis and prevented proliferation of mature T cells (34).
Functionally, the c-Myb protein can be separated into three distinct domains: a DNA-binding domain, a trans-activating domain, and a negative regulatory domain (NRD). The DNAbinding domain consists of three 51-52 amino acid imperfect repeats, each containing three conserved tryptophan residues evenly spaced (35,36). Protein NMR studies have recently confirmed that the second and third of these repeats are required for contact with the essential components of the Myb consensus binding site: the first A, C, and G nucleotides of the TAACNG sequence (37). The 50-amino acid trans-activation domain lies C-terminal to the DNA-binding domain and is hydrophilic and acidic (23), consistent with trans-activating domains of other factors. Much attention has been devoted to the negative regulatory domain present at the C terminus of the c-Myb protein. This region contains a classical leucine zipper and evidence currently suggests that when c-Myb homodimerizes via this domain, its DNA-binding and trans-activating capacity is compromised (38). At high c-Myb concentrations, dimerization is favored and DNA-binding and transactivation are ameliorated (38); Myb is therefore described as being self-limiting or self-squelching. In fact, the transforming capacity of C-terminally truncated viral Myb proteins has been shown to result from increased trans-activating capacity due to loss of this negative regulatory domain (39). The C terminus also contains a serine phosphorylation site that negatively regulates trans-activating activity. Mutation of this phosphorylation site to alanine results in a 2-7-fold increase in the trans-activating capacity of c-Myb (40). The C-terminal domain appears to serve other functions as well. Favier and Gonda (41) have shown that this domain can bind other proteins which may act either as co-activators or suppressors (38). In fact, Ness' (42) group has shown that the N and C termini of c-Myb can associate and preclude binding of the co-activator, p100. Hence, there are multiple levels at which c-Myb can be regulated via its C-terminal negative regulatory domain.
In this report, we demonstrate that c-Myb activates expression of a luciferase reporter gene under control of the human topo II␣ promoter. A role for c-Myb in topo II␣ expression appears, however, to be restricted to cells of hematopoietic origin. Use of dominant negative c-Myb inhibitors indicates that c-Myb is likely to play a major role in basal topo II␣ expression in HL-60 leukemia cells. Evidence is also presented that activation of topo II␣ expression may extend to other more widely expressed Myb family members such as B-Myb, a factor whose own expression is linked to the G 1 /S boundary of the cell cycle. This report establishes an association between topo II␣, an enzyme essential to the completion of cell division, and a transcription factor family linked intimately to tumor cell proliferation.

MATERIALS AND METHODS
Cell Culture-HL-60 human promyelocytic leukemia cells (ATCC CCL 240), U937 histiocytic leukemia cells (ATCC CRL 1593), CCRF-CEM lymphoblastic leukemia cells (ATCC 119), and HeLa S3 human cervical carcinoma cells (ATCC CCL 2.2) were obtained from the American Type Culture Collection (Rockville, MD). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . Exponentially growing suspension cultures of leukemia cells were propagated by subculturing at 5 ϫ 10 5 cells/ml every 2-3 days in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin G, and 50 g/ml streptomycin sulfate. HeLa cells were maintained in monolayer culture in Dulbecco's modified essential medium containing the same serum supplements and were subcultured at 5 ϫ 10 5 cells per 100-mm dish every 2-3 days. For all gene transfer experiments, all cells were freshly subcultured at the indicated cell densities 48 h prior to transfection.
Reporter and Expression Plasmids-All topo II␣-promoter-reporter vectors were constructed in the background of pA 3 LUC (43) as described previously (20). CMV-driven c-Myb and c-Myb⌬NRD plasmids (44) were a gift from Dr. Prem Reddy (Fels Institute, Temple Univer-sity, Philadelphia, PA). The SV40-driven c-Myb expression plasmid pMbm1 (31) was kindly provided by Dr. Edward Prochownik (Children's Hospital, Pittsburgh, PA). The dominant negative c-Myb expression plasmid (34) (pSCDMS/MenT) was a gift of Dr. Kathy Weston (Institute for Cancer Research: Royal Cancer Hospital, London). The human B-Myb expression plasmid (pKCB-myb) was generously provided by Dr. Roger Watson (Ludwig Institute for Cancer Research, London). Internal control ␤-galactosidase expression plasmids were obtained from either Dr. David Gordon (RSV-␤-gal; University of Colorado Health Sciences Center, Denver, CO) or Clontech (CMV-␤-gal). A passive, dominant negative Myb plasmid (CMV-mybDBD) was constructed by polymerase chain reaction amplification of the DNA-binding domain of c-Myb (codons 84 -212) followed by subcloning into the HindIII/BamHI site of pcDNA3 (Invitrogen, La Jolla, CA). The topo II promoter-reporter vector (Ϫ562TOP2LUC/MBSmut) was constructed by polymerase chain reaction mutagenesis of the putative Myb binding site at Ϫ16 to Ϫ11 from TAACCG to TCGACT using the 4-oligonucleotide method of Higuchi et al. (45). The fidelity of all constructs generated by polymerase chain reaction was confirmed by dideoxy DNA sequencing using Sequenase 2.0 (U. S. Biochemical Corp.). All plasmid DNA used for transfections was propagated in Escherichia coli DH5␣, then isolated by a standard alkaline-SDS lysis protocol followed by double purification on isopycnic CsCl gradients.
Transient Gene Transfection Protocol-Exponentially growing HL-60, U937, or CCRF-CEM cells were seeded as described above, then collected by centrifugation at 700 ϫ g for 5 min. The resulting cell pellet was resuspended in fresh RPMI 1640, with all supplements, at a cell density of at least 5 ϫ 10 7 cells/ml. Two-hundred microliters of this cell suspension (ϳ1 ϫ 10 7 cells) was combined with 20 g of reporter plasmid and the indicated amounts of effector plasmid, then transferred to a 0.4-cm gap width metal-lined electroporation cuvette (Invitrogen). DNA was introduced to the HL-60 cells by delivering a charge to the cell suspension using an IBI geneZAPPER electroporator set at 250 V and 950 microfarads. The observed time constant was usually within the range of 48 -56 ms. The cell suspension was then removed to a 60-mm culture dish containing 3 ml of supplemented RPMI 1640. Harvest times varied from 4 to 24 h as indicated in each figure. In general, each group contained three samples and experiments were performed 3-5 times.
For the HeLa cell experiments, the procedure was essentially the same as described above except that Dulbecco's modified essential medium was used for resuspension and plating, 1 ϫ 10 6 cells were used in each transfection, and the electroporation conditions were 180 V and 500 microfarads. Time constants generally ranged from 21 to 26 ms.
Experiments with CMV-driven expression vectors included an appropriate amount of the empty CMV vector, pcDNA3, such that each group had an equivalent total amount of CMV vectors. Controlling each group with empty vector was required since the CMV promoter alone appears to usurp general transcription factors; therefore, basal topo II␣ promoter activity varies between experiments as a result of the fact that different amounts of total CMV vectors were used in each experiment. However, the absolute magnitude of Myb-dependent trans-activation remained relatively constant regardless of the amount of CMV vectors employed.
Cell Harvest and Luciferase Assay-Cell pellets collected by centrifugation at 1000 ϫ g for 5 min were washed once with phosphatebuffered saline, then resuspended in 100 l of 100 mM potassium phosphate, pH 7.8, containing 1 mM dithiothreitol. Cells were lysed by three cycles of freezing in dry ice, thawing at 37°C for 30 -45 s, and vortexing for 15 s in order to liberate luciferase enzyme. Following the third freeze-thaw cycle lysates were centrifuged at 10,000 ϫ g for 10 min, then supernatants were removed, quantitated, and immediately assayed for luciferase activity. Luciferase activity was determined by combining 25-40 l of cell supernatant with 160 -175 l of luciferase assay buffer (100 mM potassium phosphate, pH 7.8, 1 mM dithiothreitol, 15 mM MgSO 4 , 5 mM ATP). The luciferin substrate (Analytical Bioluminescence, San Diego, CA) was prepared at a final concentration of 1 mM in 100 mM potassium phosphate, pH 7.8, 1 mM dithiothreitol, and 100 l was used to initiate each reaction. Luminescence of each reaction was indicated by arbitrary light units as quantitated over 15 s by a Los Alamos Diagnostics 535 Luminometer (Turner Designs, Mountain View, CA). Each sample was assayed twice, background was subtracted, and individual data expressed as total light units backcalculated for the entire cell sample. Group data was expressed as mean Ϯ S.E.
In most cases a plasmid containing the cytomegalovirus immediate early promoter/enhancer upstream of the E. coli lacZ gene (encoding ␤-galactosidase), denoted pCMV-␤-gal, was co-transfected (0.5-1 g) as an internal control. Preliminary experiments with B-myb expression plasmid revealed that the CMV-␤-gal construct was responsive to this factor. Therefore, experiments employing the B-myb expression plasmid used an RSV-driven ␤-gal internal control to alleviate this problem. After luciferase assay, an aliquot of cell supernatant was used for assay of ␤-gal activity using 2-nitrophenyl-␤-D-galactopyranoside as the substrate according to the method detailed by Sambrook et al. (46). E. coli ␤-galactosidase (Boehringer Mannheim) was used as the standard. Luciferase values were corrected for ␤-gal internal control activity by dividing total light units by total milliunits of ␤-gal activity.

c-Myb Expression Constructs Activate Topo II␣ Promoterreporter Expression-
The common link between c-Myb, topo II␣, and hematopoietic cell proliferation led to questioning whether c-Myb could activate the topo II␣ promoter. To test this hypothesis, a CMV-driven c-myb expression plasmid was co-transfected into HL-60 leukemia cells along with the topo II␣ promoter-luciferase reporter construct, Ϫ562TOP2LUC. As illustrated in Fig. 1, c-Myb was both a potent and efficacious activator of the topo II␣ promoter. As little as 0.2 g of c-myb expression plasmid activated reporter expression reproducibly and this effect achieved a maximum of 4.6-fold over basal levels (Fig. 1). The expression of c-myb under control of the less robust SV40 promoter (pMbm1) also activated reporter expression more than 3-fold. Interestingly, the dose-response relationship with CMV-myb exhibited an inverted U-shape, as has been described previously for other c-Myb-responsive genes (38). This diminution of trans-activation at high levels of c-myb expression is believed to occur due to the self-squelching nature of its NRD through which inhibitory protein interactions and phosphorylation can occur (38,40,42).
It would then follow that a c-Myb molecule lacking the NRD but still containing its DNA-binding and trans-activating domains would produce a more classical dose-response curve, that is, without the self-squelching plateau. To confirm this hypothesis with the topo II␣ promoter, HL-60 cells were cotransfected with Ϫ562TOP2LUC and varying amounts of plasmids driving production of either full-length c-Myb or c-Myb lacking the NRD (⌬NRD). The latter truncated c-Myb protein proved to be a far more effective trans-activator of the the topo II␣ promoter than wild-type c-Myb. As illustrated in Fig. 2, c-Myb produced a dose-response profile similar to that in Fig. 1 with a maximum of 4.7-fold activation over basal levels. In contrast, c-Myb⌬NRD resulted in 10.5-fold activation at an amount of expression plasmid where the wild-type c-Myb effect had plateaued, achieving a peak of 18.6-fold stimulation of basal topo II␣ promoter activity (Fig. 2). Specificity of both c-Myb proteins for trans-activating the topo II␣ promoter was demonstrated by the fact that neither construct activated reporter activity when under control of a heterologous herpes simplex virus thymidine kinase promoter (pT81LUC (47)) or with the promoterless luciferase parent vector, pA 3 LUC (data not shown). Taken together, these data support the suggestion that c-Myb trans-activates the topo II␣ promoter and that this trans-activation function is autoregulated via its C-terminal NRD.
Experiments presented thus far employed a 20 -24 h posttransfection harvest time. It was reasoned that a temporal assessment of c-Myb-mediated trans-activation of the topo II␣ promoter might support a direct role for c-Myb in this response, as opposed to c-Myb acting as an initiator of a cascade culminating in topo II␣ promoter activation. HL-60 cells were therefore co-transfected with Ϫ562TOP2LUC and either the control CMV empty vector or the CMV-myb effector plasmid and luciferase activity quantitated at times ranging from 4 to 18 h post-transfection. Strikingly, robust (3.2-fold) topo II␣ promoter trans-activation was observed as quickly as 4 h posttransfection (Fig. 3). Although basal luciferase expression expectedly decreases at later time points, the magnitude of transactivation remained 3-5-fold at later time points. If Myb were acting indirectly through activation of a second factor, a greater delay in appearance of trans-activation would have been expected. For example, increased expression of topo II␣ or other unrelated genes does not occur for 12-72 h after treatment with a pleiotropic activator of gene expression, the histone deacetylase inhibitor sodium butyrate (20, 48 -50). The observed rapidity of this c-Myb-mediated trans-activation is therefore consistent with a direct role for this factor in topo II␣ gene regulation.
c-Myb trans-Activation of the Topo II␣ Promoter Localizes to a Consensus Myb-binding Site-The consensus c-Myb-binding site that has been described is TAACNG, although flanking bases also appear to contribute to binding affinity (22,24,25). A match for this site exists in the human topo II␣ promoter at Ϫ16 to Ϫ11 (5Ј-TAACCG-3Ј) and this site is conserved exactly in the corresponding region of the Chinese hamster topo II␣ promoter described recently (51). A series of 5Ј-promoter deletions were constructed in the context of the luciferase reporter vector and then independently co-transfected with CMV-myb into HL-60 cells. As shown in Fig. 4, c-Myb trans-activated both a larger promoter construct which extended 5Ј from Ϫ562 (Ϫ1200) as well as minimal promoter-reporter constructs (Ϫ90 and Ϫ51) by approximately the same magnitude (3-4-fold) over basal levels. These data suggest that an element which was positively responsive to c-Myb was located within Ϫ51 of the human topo II␣ promoter, consistent with the location of the putative Myb-binding site at Ϫ16 to Ϫ11.
To directly ascertain the functionality of this putative c-Mybbinding site, the sequence TAACCG was altered by sitedirected mutagenesis to TCGACT in the context of Ϫ562TOP2LUC. As shown in Fig. 5, mutation of this site completely abrogated c-Myb-dependent trans-activation in HL-60 cells. In addition, mutation of the c-Myb site resulted in a dramatic 97% reduction in basal activity of this construct (Fig. 5). This observation appears to be unique to the topo II␣ gene since c-Myb-binding site mutations in other gene promoters does not greatly affect basal transcription levels (52,53). It is therefore possible that in the TATA-less topo II␣ promoter, the c-Myb site may overlap with transcriptional initiation sequences. Consistent with this suggestion, the Myb site lies immediately between two palandromic promoter sequences which have been suggested previously to be involved in transcriptional initiation of this gene (54). In fact, c-Myb may form a bridge with the basal transcriptional apparatus through its recently demonstrated interaction with p100, a factor known to bind TFIIE (42).
c-Myb as an Endogenous Regulator of the Topo II␣ Promoter in HL-60 Cells-Although mutation of the topo II␣ promoter c-Myb-binding site greatly reduced basal reporter expression from this construct, its proximity to the transcriptional initiation site confounds our understanding of the relative contribution of c-Myb to basal topo II␣ expression in human leukemia cells. A question which therefore remained was whether c-Myb is an endogenous modulator of topo II␣ gene expression in myeloid cells. The antisense oligonucleotide approach has been taken previously to ameliorate candidate c-Myb-dependent responses (30). In preliminary experiments, an antisense c-Myb expression vector co-transfected into HL-60 cells did indeed cause a dose-dependent attenuation of topo II␣ promoterdriven luciferase activity (data not shown). However, a scrambled control expression vector had a similar, but non-sequencespecific effect. This data may be reconciled by the recent demonstration of a non-antisense mechanism for antisense c-Myb oligonucleotides (55). These sequences appear to exert an antiproliferative effect via a "G quartet" sequence contained within, possibly by sequestering basic fibroblast growth factor when administered extracellularly (56). Therefore, these results indicated an alternative approach would be necessary to inactivate endogenous c-Myb.
Weston's group has recently constructed an elegant chimeric expression vector which encodes the c-Myb DNA-binding domain fused with the Drosophila engrailed transcriptional repressor domain (34). When the plasmid is introduced into cells the resulting protein competes for binding of Myb consensus sites with endogenous c-Myb, repressing rather than activating transcription. This chimera was demonstrated to be effective both in transfection assays as well as in T-cell specific gene expression in transgenic mice (34). We therefore obtained this dominant negative expression construct (pSCDMS/MenT) for co-transfection of HL-60 cells with Ϫ562TOP2LUC. A second, passive, dominant negative competitor of c-Myb binding was also employed which consisted only of the c-Myb DNA-binding domain (pCMV-mybDBD). To first demonstrate the effectiveness of this approach, the dominant negative constructs were tested for their ability to block both c-Myb or c-Myb⌬NRDmediated trans-activation of the topo II␣ promoter. As illus- The putative c-Myb-binding site (MBS) at Ϫ16 to Ϫ11 was mutated to TCGACT (mutated bases underlined) in the context of Ϫ562TOP2LUC as described under "Materials and Methods." HL-60 cells were co-transfected as described previously with 20 g of either Ϫ562TOP2LUC (wild-type MBS) or Ϫ562TOP2LUC/MBSmut (mutant MBS) and 1 g of either empty CMV vector or CMV-myb. Cells were harvested at 24 h and processed for measurement of each reporter enzyme activity. The ␤-gal reference plasmid was not included in this experiment due to the already low activity of the Ϫ562TOP2LUC/MBSmut construct. Numbers above each bar represent the -fold luciferase activity relative to the basal activity of each respective construct in the absence of any effector plasmids. u, CMV only; s, CMV-myb. trated in Fig. 6A, the passive and active c-Myb dominant negative peptides effectively reduced c-Myb-stimulated promoter activity to ϳ120 and 30% of basal levels, respectively, whether wild-type or truncated c-Myb was used as the activator. This finding confirmed that the dominant-negative Myb proteins could attenutate the effects of exogenously-derived Myb. But most importantly, the dominant negative peptides also reduced basal topo II␣ promoter activity to 60% (for mybDBD) and 38% (pSCDMS/MenT) of control (CMV only control group, Fig. 6A). This effect was specific for the topo II␣ promoter since the dominant negative Myb constructs had no effect on the internal control ␤-galactosidase expression plasmid. This finding suggests that c-Myb, or perhaps a related family member, plays a central role in topo II␣ expression in proliferating HL-60 leukemia cells.
The specificity of the more efficacious dominant negative construct, pSCDMS/MenT, was addressed by comparing its dose-response relationship in HL-60 cells versus HeLa cells, a human cervical carcinoma line known to lack c-Myb. In Fig. 6B, the dominant negative construct elicited a reproducible suppression of topo II␣ promoter activity in HL-60 cells with as little as 0.2 g of plasmid, and further suppression was dosedependent. In contrast, up to 2 g of pSCDMS/MenT had no suppressive effect and even slightly stimulated the topo II␣ promoter-reporter when co-transfected into HeLa cells. Therefore, the specificity of this dominant negative Myb construct for endogenous c-Myb in HL-60 cells was confirmed by its lack of suppressive effect in a c-Myb-negative cell line (Fig. 6B). Furthermore, this lack of effect in HeLa cells also suggests that the Myb dominant negative protein was not acting in HL-60 cells simply by physical blockade of transcriptional initiation.
Cell Type-specific c-Myb trans-Activation of Topo II␣ Promoter-We have also begun to investigate the cell type-specific trans-activation by c-Myb of topo II␣ promoter-reporter constructs in other hematopoietic or lymphoid cells. While c-Myb is normally expressed in immature cells of these lineages, only a subset of these cells are capable of supporting trans-activation in the case of one c-Myb target gene, mim-1 (57). Ness et al. (58) demonstrated that cell-specific trans-activation by c-Myb was dependent on at least one differentially expressed factor, NF-M, which acts in a bipartite fashion with c-Myb. It is clear that c-Myb alone is not capable of substantial trans-activation in either epithelial cells or fibroblasts (53,58), presumably due to lack of a combinatorial activator like NF-M or other recently characterized Myb co-activators (42,59). Therefore, co-transfections with CMV-myb and Ϫ562TOP2LUC were performed with three other human tumor cell lines: U937 histiocytic leukemia cells, CCRF-CEM lymphoblastic pre-T cell leukemia, and HeLa epithelial cervical carcinoma cells. As depicted in Fig. 7, both the U937 and CCRF-CEM cells could support substantial c-Myb-mediated trans-activation of the topo II␣ promoter-reporter construct. While CCRF-CEM cells were somewhat less inducible than U937 cells, it should be recognized that CCRF-CEM cells already possess the highest basal topo II␣ promoter activity of any cell line tested to date; therefore, other cooperating factors may already be in near-limiting quantities. In contrast, c-Myb was less effective in stimulating topo II␣ transcription in HeLa cells, especially with high amounts of c-Myb expression plasmid. These results suggest that c-Myb regulation of topo II␣ is cell-type specific, consistent with other previously described Myb target genes.
B-Myb Also trans-Activates the Human Topo II␣ Promoter-In almost every cell type tested, topo II␣ abundance is directly proportional to proliferation rate or growth fraction. Since c-Myb production is restricted to hematopoietic cells with a few exceptions (60), it seems unlikely that this factor is responsible for the proliferation-dependent regulation of topo II␣ observed in numerous other cell types. Other Myb-related factors, especially B-Myb, appear to be more widely expressed in other tissues (61), including human myeloid cells (62). B-Myb is also intimately associated with cell cycle progression. Of note, B-myb transcriptional repression in G 0 and early G 1 phase and activation at the G 1 /S boundary are associated with two distinct classes of E2F-containing DNA binding complexes (63). Recent work also suggests the p107 retinoblastoma family member may exert its growth-suppressive activity by repressing B-myb transcription, perhaps in a complex with an E2F protein (64). With this link to cellular growth control, B-Myb was therefore evaluated as a potential activator of the topo II␣ promoter in both a myeloid and non-myeloid cell line.
When a CMV-driven B-myb expression vector (pKCB-myb; gift of Dr. R. J. Watson) was co-transfected with Ϫ562TOPLUC into HL-60 cells, robust increases in reporter activity were observed (Fig. 8). While this activation required greater amounts of B-myb expression plasmid than with c-myb plasmids (Fig. 1), maximal promoter activation achieved a far greater magnitude (15-fold at 15 g) and a plateau was not observed (Fig. 8) trans-activated the topo II␣ promoter construct in HeLa cervical carcinoma cells (Fig. 8). This observation is consistent with the fact that in HeLa cells, B-Myb has been shown to be an effective trans-activator of other c-Myb-responsive genes (65). It is therefore conceivable that other more widely expressed Myb family members like B-Myb serve to activate topo II␣ expression in non-hematopoietic cell types.

DISCUSSION
This report provides evidence for trans-activation of the topo II␣ promoter by a transcription factor family well known to play a role in cellular proliferation. Based on its catalytic role essential for chromosomal segregation, topo II␣ is a logical downstream effector for the growth-stimulatory effects of the Myb transcription factor family. Activation of the topo II␣ promoter by c-Myb was comparable to that described for other c-Myb-responsive genes in that, 1) an inverted U-shaped doseresponse relationship was observed for wild-type c-Myb; 2) a C-terminal-truncated c-Myb protein lacking the NRD led to more robust trans-activation; 3) c-Myb-mediated trans-activation occurred rapidly following transfection; 4) c-Myb-mediated trans-activation was selective for hematopoietic cells; 5) mutation of the c-Myb-binding site abrogated c-Myb-mediated transactivation; and 6) a dominant negative c-Myb protein was capable of attentuating trans-activation by either c-Myb or c-Myb⌬NRD.
Of greatest relevance is the fact that the dominant negative c-Myb proteins were able to block 40 -60% of the basal activity of a topo II␣ promoter-reporter construct in HL-60 cells. Specifically, the fact that the passive c-Myb DBD competitor reduced basal promoter activity supports an endogenous role for c-Myb in topo II␣ expression in HL-60 cells. The possibility has been considered that the dominant negative proteins simply prevented assembly of the basal transcription apparatus since an intact Myb site also appeared to be essential for the majority of basal topo II␣ expression (Fig. 5). However, the more active of the two c-Myb dominant negative proteins had no effect or slightly stimulated topo II␣ promoter activity in HeLa cells (Fig. 6B). This finding suggests that the dominant negative proteins acted in HL-60 cells by precluding c-Myb binding and not via general blockade of transcriptional initiation. Taken together, the mutagenesis and dominant negative experiments support a role for endogenous c-Myb in topo II␣ expression in HL-60 cells.
The significance of this finding extends beyond basic studies of transcriptional regulation. The transcriptional basis of topo II␣ regulation has also been investigated as a mechanism of tumor cell resistance to drugs which trap topo II␣ in covalent complexes with DNA (66). For example Sp3, an Sp1 family member which acts as a transcriptional repressor, is believed to bind an Sp1 site in the human topo II␣ promoter (67). In topo II-drug-resistant carcinoma cell lines which underexpress topo II␣, resistance appears to be due to overexpression of the Sp3 repressor (67). With Myb proteins appearing responsible for the bulk of basal topo II␣ promoter activity in HL-60 cells, altered Myb regulation should also be investigated as another basis for topo II-drug resistance in leukemias.
While dominant negative Myb proteins also clearly abrogate trans-activation by ectopically expressed c-Myb proteins, it is not yet known whether the function of other Myb family members is also inactivated by these inhibitors. This point will be particularly important to investigate further since B-Myb, which is also produced by HL-60 cells (62), does not always bind to and trans-activate through the same element as c-Myb (24,68). In fact, the lack of effect of the Myb/engrailed chimera in HeLa cells (Fig. 6B), which are known to produce B-Myb, suggests that B-Myb may not be acting through the c-Myb site at Ϫ16 to Ϫ11 of this promoter.
This report also carries implications for understanding the role of c-Myb in cell proliferation. While a pivotal role for c-Myb in hematopoietic cell growth has been appreciated, few c-Myb target genes are known which trigger or facilitate proliferation. For example, c-Myb is known to trans-activate the cdc2 kinase gene (52) but its role in trans-activating the DNA polymerase ␣ gene remains equivocal (69). The present report therefore implicates topo II␣, an enzyme essential for chromosomal segregation prior to mitosis, as another gene target for the prolifer -FIG. 7. c-Myb trans-activation of ؊562TOP2LUC in leukemia and epithelial cell lines. The T-cell leukemia (CCRF-CEM), promonocytic leukemia (U937), and cervical epithelial carcinoma (HeLa) lines were independently co-transfected with Ϫ562TOP2LUC (20 g), CMV-␤-gal reference plasmid (1 g), and the indicated amounts of CMV-myb expression plasmid. The control group in each case (0 g of CMV-myb) received 2 g of the empty CMV vector. Cells were harvested at 24 h and processed for measurement of each reporter enzyme activity. Numbers above each bar represent the -fold increase over each respective CMV only control.

FIG. 8. Topo II␣ promoter trans-activation by B-Myb. HL-60
and HeLa cells were independently co-transfected with Ϫ562TOP2LUC (20 g), RSV-␤-gal reference plasmid (1 g), and the indicated amounts of a CMV-B-myb expression plasmid (pKCB-myb). A ␤-gal internal control driven by the RSV promoter was required since the standard CMV-␤-gal control was activated by B-Myb. The control group in each case received 15 g of the empty CMV vector and each B-Myb group received an amount of CMV vector to bring the total to 15 g. Cells were harvested at 24 h and processed for measurement of each reporter enzyme activity. Numbers above each bar represent the -fold increase over each respective CMV only control. ative effects of c-Myb at least in hematopoietic cells.
Conversely, the self-squelching property of c-Myb via its NRD presents this factor as a rather attractive candidate for regulation of topo II␣. Topo II␣ levels rarely vary by more than 2-or 3-fold in proliferating cells in culture (70 -72). In fact, attempts at overexpression of recombinant topo II␣ in a variety of hosts have been difficult and often require conditional expression systems (73). Stringent control of normal, cellular topo II␣ expression therefore logically requires a proliferation-dependent transcriptional program that itself is either tightly regulated, or is intrinsically self-regulating. The outcome of our experiments with c-Myb begins to establish a causal relationship for this logic. How this hypothesis might extend to topo II␣ regulation in non-hematopoietic tissues is somewhat less clear. We have begun to investigate the role of other Myb family members, like B-myb, which are more widely expressed in different tissues (61). But unlike c-Myb, the C terminus of B-Myb does not act in a negative regulatory fashion (74). Instead, the B-Myb C terminus is believed to bind co-activating proteins whose abundance appears to limit its efficacy as a trans-activator (65). Based on this finding, it is surprising that the trans-activating effect of B-Myb did not appear to be limiting in transfection studies (Fig. 7). Nonetheless, B-myb expression is tightly regulated at the G 1 /S boundary (63) and precedes the known increase in topo II␣ expression in G 2 /M (71,72). The results in Fig. 8 make it tempting to speculate that the Myb family of transcription factors might play a global role in topo II␣ regulation, but investigation of B-Myb effects in a more extensive panel of non-hematopoietic cells is warranted.