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[Cancer Research 60, 121-128, January 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

ICBP90, a Novel Human CCAAT Binding Protein, Involved in the Regulation of Topoisomerase II{alpha} Expression1

Raphaël Hopfner, Marc Mousli, Jean-Marc Jeltsch, Angélique Voulgaris, Yves Lutz, Cristi Marin, Jean-Pierre Bellocq, Pierre Oudet and Christian Bronner2

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, 67404 Illkirch Cedex, Communauté Urbaine de Strasbourg, France [R. H., M. M., A. V., Y. L., P. O., C. B.]; INSERM U425, Faculté de Pharmacie, 67401 Illkirch Cedex, France [M. M., C. B.]; UPRES1329, ESBS, 67400 Illkirch Cedex, France [J-M. J.]; and Laboratoire d‘Anatomologie-Pathologie, Hôpital Universitaire de Hautepierre, 67200 Strasbourg, France [C. M., J-P. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase II{alpha} gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase II{alpha} and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase II{alpha}. Immunohistochemistry experiments showed that topoisomerase II{alpha} and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase II{alpha} expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic DNA topoisomerases are ubiquitous nuclear enzymes that alter the topology of DNA. Topoisomerase I and TopoII3 introduce in DNA transient single- and double-stranded breaks, respectively (1 , 2) . These mechanisms are required during the cell cycle for DNA replication, chromosome condensation, and segregation (1, 2, 3, 4) . Human topoisomerase I is encoded by a single-copy gene mapped to chromosome region 20q12–13.2 (5) . Two types of human TopoII have been identified and mapped to different chromosomes (17q21–22 for TopoII{alpha} and 3p24 for TopoIIß; Refs. 6, 7, 8, 9 ). The structure and conformational changes of the {alpha} isoform, which are required for its activity, have been recently visualized in our laboratory by electron microscopy (10) . During interphase, the {alpha} isoform (170 kDa) has a nuclear location and is found in both the nucleoplasm and the nucleoli, whereas the location of the ß form (180 kDa) was harder to establish (Ref. 11 and references therein). During mitosis, the {alpha} isoform is found associated with metaphase chromosomes with a uniform distribution axially located on the arms of the chromosomes and on the centromeres, whereas the ß form is predominantly cytoplasmic, with a small amount detected in metaphase scaffolds (Ref. 11 and references therein).

TopoII{alpha} appears to be the preferential, primary target for anticancer drugs, such as etoposide, teniposide, or amsacrine (3) . TopoII{alpha} expression is cell-cycle regulated, whereas that of the ß isoform is relatively constant throughout the cell cycle (11 , 12) , except in an amsacrine-resistant leukemia cell line with mutant TopoII{alpha} in which the absence of TopoIIß was reported (13) . The molecular mechanisms involved in the variations in TopoII{alpha} expression depend on the cell type. For example, in HeLa cells, the expression of TopoII{alpha} increases by 3-fold in the late S phase and G2-M (14) . This increase is paralleled by a 15-fold elevation in mRNA levels in the late S phase, which is in part due to a 2-fold increase in the level of transcription and an 8-fold increase in mRNA stability (14) . In contrast, in etoposide/teniposide-resistant human epidermoid cell lines (KB/VP-2 and KB/VM-4 cells), an increase in transcription has been suggested to be mainly involved in the expression of TopoII{alpha} during the G2-M phases (15) . The decrease in TopoII{alpha} expression as the cell progresses from mitosis to the G1 phase has been shown in chicken lymphoblastoid cells to result from protein degradation (16) . The endogenous and exogenous factors responsible for changes in TopoII{alpha} expression are numerous and variable, including oncogenes, heat shock, and exposure to cytotoxic drugs (12) such as topotecan, an anti-TopoI drug (17) . Differential expression of TopoII{alpha} according to the tumor tissue and to the cell type has been reported. For instance, small cell lung carcinoma nuclei exhibit higher levels of TopoII{alpha} than nuclei from non-small cell lung carcinoma (18) . Also, a small cell lung cancer cell line (H209/V6) selected for its resistance to etoposide showed lower TopoII{alpha} expression than the parental cell line (19) . The factors that control levels of TopoII{alpha} and TopoIIß isoforms are potentially of interest in the context of drug resistance (20) . In this context, identifying new transcription factors acting on the promoter of the TopoII{alpha} gene may be of particular importance, considering that the transcription factors known today cannot fully explain the variation of TopoII{alpha} expression in physiological or pathophysiological situations (12 , 20) .

Human (21) , Chinese hamster (22) , and rat (23) TopoII{alpha} gene promoters have been characterized. Between nucleotide -617 and the transcription start site, five CCAAT boxes in the inverted position called ICB (ICB1 to ICB5), one ATF binding sequence, and two GC-rich boxes are found for the human promoter. Some transcription factors interacting with the promoter of the human TopoII{alpha} gene such as c-myb (24) , p53 (25) , ATF (26) , Sp1, and Sp3 (18 , 27) have been proposed. Isaacs and coworkers (28) identified NF-Y (also called CBF, ACF, and CP1) as a component of a proliferation-induced complex that binds in vitro to the critical ICB2 in the promoter of the TopoII{alpha} gene, although NF-Y is still detectable in confluence-arrested cells. However, apart from NF-Y, proteins or complexes acting on the ICBs in the promoter of the human TopoII{alpha} gene are not yet all identified and characterized. For instance, two proteins with estimated molecular weights of 140 kDa and 90 kDa have been shown to bind ICB1 to ICB4 and ICB5 of the human TopoII{alpha} gene promoter, respectively (29) . Based on these observations, we hypothesized that beside NF-Y, unknown CCAAT binding proteins may participate in the regulation of the TopoII{alpha} gene expression. To identify one of these putative proteins, we used the one-hybrid system that allows the isolation of cDNA clones encoding sequence-specific DNA-binding proteins. This method allows access to novel proteins with some indications of their properties and therefore of their putative roles. In the present study, we identified a novel human 793-amino-acid-long protein (89,758 Da) that exhibited ICB binding properties. This protein, called ICBP90, participates in TopoII{alpha} expression and is preferentially expressed in proliferating cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The One-Hybrid System for the Cloning of CCAAT Binding Proteins.
The one-hybrid system is a powerful technique, which allows one to detect in vivo in yeast the interaction of proteins with specific DNA sequences by screening cDNA libraries. Subsequently, direct access to the corresponding cDNA of the bound protein is possible. Several studies have reported finding new proteins with this method, in which the protocols are well-described (30 , 31) . Briefly, the oligonucleotides, 5'-AATTCGATTGGTTCTGATTGGTTCTGATTGGTTCTT-3' and 5'-CTAGAAGAACCAATCAGAACCAATCAGAACCAATCG-3', were synthesized and annealed. As instructed by the manufacturer (Clontech, Palo Alto, CA), the target-reporter construct had three tandem copies of ICB2 (ICB2 x 3). As shown above, one copy is italicized and the CCAAT boxes are in bold. To examine the specificity of protein binding to the ICB, the following oligonucleotides, which contain three tandem copies of the GC1 (GC1 x 3) box present in the promoter, were synthesized and annealed: 5'-AATTCGGGGCGGGGCCGGGGCGGGGCCGGGGCGGGGCT-3' and 5'-CTAGAGCCCCGCCCCGGCCCCGCCCCGGCCCCGCCCCGG-3'. The resulting target DNA fragments were cloned in the polylinker of the integrative plasmid pHISi-1 (Clontech) by cohesive end ligation at the EcoRI and XbaI sites, upstream of the minimal promoter of the his3 gene. The yeast strain YM4271 (Clontech) was used for transformation, and yeast clones that have integrated the plasmid into their genome were selected on Synthetic Dropout medium lacking histidine. Two clones were kept, one for ICB2 and one for the GC1 box. The plasmid construct and its integration into the yeast genome were analyzed by PCR.

A cDNA library of the Jurkat cell line, cloned into the EcoRI site of the polylinker downstream of the GAL4-AD of pGAD10 vector (Clontech), was used for screening according to the instructions of the manufacturer. Positive clones were grown on a selective medium lacking histidine and leucine. Plasmid DNA from these clones were rescued and introduced into Escherichia coli XL1-blue by electroporation. Sequencing of the inserts was performed in our laboratory (Service d’Adrien Staub) on a plasmid DNA template, which was purified from 1.5 ml of culture using a minipreparation kit (Bio-Rad, Hercules, CA). A 5'-stretched human thymus {lambda}gt10 cDNA library (Clontech) was screened by plaque hybridization to recover the full-length cDNA encoding the protein. For this screening, we used the same 679-bp length cDNA probe as for the human multiple tissue RNA dot blot (see later).

Human Multiple Tissue RNA Dot Blot Analysis.
A 679-bp-long cDNA probe corresponding to amino acids 269 to 500 of ICBP90 was synthesized by the PCR with Taq polymerase (Sigma, St. Louis, MO). The probe was labeled by random priming with dCTP-{alpha} 32P and purified on G50-Sephadex columns (Pharmacia, Uppsala, Sweden). A multiple tissue RNA dot blot of poly(A)+ RNA from 50 different human tissues was used under high stringency for a 20-h hybridization in ExpressHyb (Clontech) at 68°C with the 32P-labeled probe. High-stringency washes were performed in 0.1x SSC plus 0.1% SDS at 68°C (32) .

Overexpression and Purification of hRS4, hRS12, ICBP59, and ICBP90.
The cDNAs, encoding hRS4, hRS12, and ICBP59, were obtained by digestion with EcoRI of the positive clones obtained in the pGAD10 vector. The cDNAs were cloned into the EcoRI site of the pGEX-4T-1 vector (Pharmacia), and the resulting recombinant DNAs were transferred into E. coli (strain BL21). Five hundred milliliters of culture of the selected clone were used when the optical density of 0.5 was reached. Overexpression was induced by isopropyl-1-thio-ß-D-galactopyranoside (1 mM) for 2 h at 37°C. The glutathione-S-transferase fusion proteins were purified using glutathione-Sepharose beads (Pharmacia) followed by an overnight cleavage with thrombin (0.05 units/ml) at 4°C (Pharmacia). The cDNA of ICBP90 (2379 bp) was synthesized by PCR using Deep Vent DNA polymerase (New England Biolabs, Beverly, MA) and oligonucleotides flanked with the EcoRI restriction site. The product of the reaction was further cloned into pGEX-4T-1 (Pharmacia) for expression of the glutathione-S-transferase fusion protein in E. coli (strain BL21). Overexpression was induced by isopropyl-1-thio-ß-D-galactopyranoside (1 mM) for 4 h at 25°C. ICBP90 was purified as described above.

Antibody Synthesis.
mAbs were synthesized in our laboratory by injection in mice of the COOH-terminal part of ICBP90 (ICBP59) starting from amino acid D263 by a standard method (33) . Two mAbs, 1RC1C-10 and 1RC1H-12, were selected for their performance in detecting ICBP90 in both Western blotting, immunocytochemistry and immunohistochemistry experiments.

Cell Cultures and Western Blotting.
HeLa cells and COS-1 cells were cultured as previously described (33, 34, 35) . Human lung fibroblasts in primary culture were prepared and cultured in DMEM/F12 supplemented with 10% FCS as described elsewhere (36) . Proliferating HeLa cells and human lung fibroblasts were harvested when 60 to 70% confluence was reached. Confluent HeLa cells and human lung fibroblasts cells were harvested at 100% confluence followed by a further incubation of 48 h in the absence of FCS. Crude cell lysates were prepared by harvesting the cells in phosphate buffer saline followed by sonication. For immunoblotting, total proteins from cell lysates were loaded for one-dimensional electrophoresis on SDS-8% polyacrylamide gels. Proteins were blotted onto nitrocellulose membranes, blocked with 10% blocking reagent (Roche Diagnostics, Mannheim, Germany), and incubated with the purified mAb (1RC1C-10) or with a mouse anti-TopoII{alpha} mAb (Roche Diagnostics), both at the concentration of 0.5 µg/ml. A sheep antimouse immunoglobulin-alkaline phosphatase (Fab fragments, Roche Diagnostics) was used at a dilution of 1:2500. For the detection of actin, a rabbit antiactin polyclonal antibody (Sigma) was used at a dilution of 1:1000. A sheep antirabbit IgG-alkaline phosphatase (Roche Diagnostics) was used at a dilution of 1:1000. Signals were detected using 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate as the substrate. All protein concentrations indicated in the figure legends were determined by the Bradford method (Bio-Rad Protein assay).

Transfection of COS-1 Cells with the pSG5 Plasmid Encoding ICBP90.
COS-1 cells were transfected with pSG5 vector (Stratagene, La Jolla, CA) in which the cDNA of ICBP90 (2379 bp) was subcloned into the EcoRI restriction site. The cDNA was synthesized by PCR using Deep Vent (New England Biolabs) and oligonucleotides flanked with the EcoRI restriction site. The plasmid construct was confirmed by nucleotide sequencing. The transfection procedure using FuGENE6 (Roche Diagnostics) was performed according to the manufacturer’s instructions with a FuGENE:DNA ratio of 3:1 (µl/µg). After transfection, cells were grown for 24 h, scraped, and collected in phosphate buffer saline as described above.

Immunocytochemistry and Immunohistochemistry.
Immunofluorescence staining of HeLa cells and transfected COS-1 cells were carried out as described elsewhere (33) , with the 1RC1C-10 and the 1RC1H-12 mAbs, respectively. The 1RC1H-12 mAb was used for COS-1 cells instead of the 1RC1C-10 mAb because it did not label the endogenous ICBP90 of COS-1 cells. For immunohistochemistry, indirect immunoperoxidase stainings of ICBP90 and TopoII{alpha} were carried out as described elsewhere (37 , 38) . Tissues from human appendixes and primary, high-grade or low-grade breast carcinomas were embedded in paraffin and fixed in 10% buffered formalin (Sigma). Serial histological sections (3 µm) were incubated overnight at room temperature with the 1RC1C-10 mAb or with an anti-TopoII{alpha} mAb (NeoMarkers, Union City, CA), and specifically bound antibodies were visualized by a Streptavidin-Biotin complex (LAB/LSAB method, Dako LSAB2 System Kit, DAKO, Carpinteria, CA).

Electrophoretic Mobility Shift Assays.
To test the ability of ICBP90 to bind specifically to ICB2, the following oligonucleotides were synthesized, 32P-end labeled using T4 polynucleotide kinase (New England Biolabs) and [{gamma} 32P]ATP (160 mCi/mmol, ICN Irvine, CA), and then annealed: 5'-ATAAAAGGCAAGCTACGATTGGTTCTTCTGGACGGAGAC-3' and 5'-GTCTCCGTCCAGAAGAACCAATCGTAGCTTGCCTTTTAT-3'.

Purified ICBP90 (1 µg) was incubated with 1 ng of 32P-end labeled oligonucleotides in 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 4 mM Tris-HCl (pH 7.9), 0.6 mM DTT, and 1 ng of poly(dI/dC) in 20 µl (30) . After 30 min incubation at room temperature, reaction mixtures were loaded on 6% polyacrylamide gels. For competition experiments, the indicated amounts of unlabeled oligonucleotides were added together with the radiolabeled probe 5 min after the addition of ICBP90. The specificity of the binding was examined using nonlabeled oligonucleotide containing a mutated ICB (ICB2m), that were subsequently annealed, (mutated bases are in bold): 5'-ATAAAAGGCAAGCTACGATTCCTTCTTCTGGACGGAGAC-3' and 5'-GTCTCCGTCCAGAAGAAGGAATCGTAGCTTGCCTTTTAT-3'.

Bacic Local Alignment Search Tool (BLAST) Searches and Domain Prediction.
Online BLAST searches were performed via the National Center for Biotechnology Information at the NIH, Bethesda, MD. SCANPROSITE and PROFILESCAN (Infobiogen, Villejuif, France) were used for protein analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selection of Clones Encoding CCAAT Binding Proteins.
Screening of the Jurkat cDNA library for putative ICB2 binding proteins using the one-hybrid system resulted in 10 clones (numbered from C1 to C10). After retransformation of the yeast strain with the corresponding isolated plasmids, only four clones (C4, C6, C8, and C10) were able to grow on the selective medium lacking histidine. The plasmids of these clones were recovered and 500 bp of the inserts were sequenced and analyzed by BLAST searches. Clones C4 and C10 encoded the ribosomal proteins hRS4 and hRS12, respectively. Clone C6 encoded a serine/threonine kinase (STPLK-1). No matches were obtained for clone C8. Clone C6 was discarded because we did not find any indication in the literature that STPLK-1 is a nuclear and/or DNA-binding protein. When the ICB2 was substituted in vivo by a GC1 box, C4, C8, and C10 were unable to activate the one-hybrid system, i.e., the his3 gene. This result indicates that the in vivo effect of these clones is dependent upon the presence of the ICB2 in the minimal promoter of the his3 gene. Clones C4, C8, and C10 were checked for their ability to bind in vitro to ICB2. The cDNA of these clones were subcloned into the pGEX-4T-1 vector, and the corresponding proteins were overexpressed in E. coli and purified. Electrophoretic mobility shift assays were then performed. Clone C8 was found to be able to bind to ICB2 (results not illustrated). This binding was competed by nonlabeled oligonucleotide containing the ICB2 but not with nonlabeled oligonucleotide containing a GC-rich box (not shown). No binding was detected with hRS4 and hRS12 (not shown). Together, these results show that only clone C8 exhibits in vitro- and in vivo-specific CCAAT box binding properties. Consequently, subsequent investigations were focused on clone C8.

Identification of the Clone C8.
The cDNA insert length of 3200 bp of C8 encoded for a 59-kDa protein that we called ICBP59 for Inverted CCAAT box Binding Protein of 59 kDa. Because no satisfactory ATG was observed, we considered that the N-terminal part of the protein was missing. Consequently, we examined the tissue distribution of C8 mRNA to subsequently use an adequate 5'-stretched cDNA library. A RNA dot blot containing 50 different human tissues was used and hybridized with a 679-bp-long cDNA probe. As shown in Fig. 1Citation , adult thymus, fetal thymus, fetal liver, and bone marrow are the richest tissues for the mRNA of C8. To a lesser extent, testis, lung, heart, and fetal kidney also show significant hybridization with the probe. In contrast, highly differentiated tissues or cells, such as the central nervous systems or peripheral leukocytes, did not show any detectable signal (Fig. 1)Citation . Consequently, to obtain the full length of C8, a 5'-stretched human thymus {lambda}gt10 cDNA library was screened by plaque hybridization. The screening of 3 x 106 clones, with the 679-bp-long cDNA probe, yielded six clones with inserts of different lengths, which have all been sequenced. Four clones, cb5, cb8, cb9, and cb10, contained an open reading frame with a potential start codon (ATG) partially consistent with Kozak’s rule and a stop codon. This resulted in a complete protein of 793 amino acids that we called ICBP90 for Inverted CCAAT box Binding Protein of 90 kDa.



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  		Fig. 1. The tissue distribution of ICBP90 mRNA. RNA dot blot was hybridized overnight with the radiolabeled 679-bp cDNA probe using ExpressHyb hybridization solution. The blot was washed and exposed on X-ray film for 1 week at -80°C with amplifying screens.

 
Features of ICBP90.
The ICBP90 protein has a predicted molecular mass of 89,758 with a isoelectric point of 7.77 based on its deduced amino acid sequence4 (Fig. 2)Citation . Two nuclear localization signals between R581-K600 and K648-K670, a zinc finger of the ring finger type (C724 to R763), a zinc finger of the PHD finger type (N310 to D366), and several putative phosphorylation sites for cAMP/cGMP-dependent kinase (4 sites) protein kinase C (7 sites), casein kinase II (8 sites), and tyrosine kinase (1 site) were found (Fig. 2)Citation . An ubiquitin-like domain, partially overlapped by a leucine zipper domain, is predicted from M1 to Q75. A consensus Rb-binding sequence (IXCXE) was found at position I725 to E729. A BLAST search for proteins homologous to ICBP90 resulted in 11 matches. Except for the mouse Np95, the homology was restricted to the PHD finger (Fig. 3)Citation and the ubiquitin-like domain (not shown). Otherwise, no homology was found other than identical anonymous Expressed Sequence Tag (accession numbers: AI084125, AI083773, AA744240, AA811055, AA806567).



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  		Fig. 2. Structural organization of ICBP90. Ubiquitin domain (white box), a zinc finger domain (PHD finger of the C4HC3 type; dotted line box), and a ring finger (C3HC4 type; black box). A putative leucine zipper domain is shown by bold amino acids. Two potential nuclear localization signals are underlined. A tyrosine kinase phosphorylation site (====), cAMP/cGMP-dependent protein kinase sites (overlined), and phosphorylation sites for casein kinase II (++++) and protein kinase C (****) were found.

 


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  		Fig. 3. Alignment of the PHD zinc finger domain of ICBP90 with other proteins found by BLAST searches. Shaded boxes, the residues conserved in at least 90% of the matched proteins; clear boxes, the residues conserved in some proteins.

 
Nuclear Localization of Endogenous and Overexpressed ICBP90.
Fig. 4ACitation shows that the endogenous ICBP90 of HeLa cells is a nuclear protein because immunostaining with 1RC1C-10 was restricted to the nucleus. To examine if the cDNA of ICBP90 (2379 bp) encodes in vivo for a nuclear protein, the cDNA was cloned into the pSG5 vector and introduced into COS-1 cells for immunostaining studies. Using the 1RC1H-12 mAb, the signal was restricted to the nucleus (Fig. 4B)Citation . Only the nucleus of transfected cells was labeled (Compare Fig. 4BCitation with Fig. 4CCitation ). This result shows that the cloned cDNA of ICBP90 led to the expression of a protein containing at least one nuclear localization signal.



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  		Fig. 4. Nuclear localization of the endogenous and overexpressed ICBP90. A, nontransfected HeLa cells were examined for the endogenous expression of ICBP90 with the 1RC1C-10 mAb. B and C, COS-1 cells were transfected with the cDNA of ICBP90 cloned into the pSG5 vector. The immunostaining signal with the 1RC1H-12 mAb (B) was restricted to transfected cells as compared to cells labeled with Hoechst 33258 (C). CY3-conjugated antimouse antibody was used as the secondary antibody at a final dilution of 1:200.

 
Differential Expression of ICBP90 and TopoII{alpha} during Cell Proliferation.
Fig. 5ACitation shows the expression of ICBP90 and TopoII{alpha} in HeLa cells and in primary cultured human lung fibroblasts at confluence or in proliferation. Proliferating and nonproliferating cells were harvested as described in the "Materials and Methods" section. In confluent HeLa cells (Lane 1), one major band was seen at 97 kDa, and a minor band was seen around 50 kDa. In proliferating HeLa cells (Lane 2), beside the 97 kDa protein, several additional bands were observed with apparent sizes of 85, 50, 46, and 44 kDa. The bands at 97 and 85 are doubled. In confluent human lung fibroblasts, a very weak expression of the 97-kDa protein can be seen (Lane 3). Conversely, the proliferation of human lung fibroblasts (Lane 4) is accompanied by an enhanced expression of the 97-kDa protein, which also appeared to be doubled. The expression pattern of ICBP90 in proliferating Jurkat, MOLT-4, and HL60 cells (data not shown) is similar to that of proliferating HeLa cells. Fig. 5BCitation shows the expression of TopoII{alpha} in HeLa cells and in human lung fibroblasts at confluence or in proliferation. In confluent and proliferating HeLa cells (Lanes 1 and 2), TopoII{alpha} expression was detected at a similar magnitude, perhaps a little bit weaker in confluent cells. In human lung fibroblasts, TopoII{alpha} was not detected in confluent cells (Lane 3) but only in proliferating cells (Lane 4). The expression pattern of TopoII{alpha} and ICBP90 in primary human bronchial smooth muscle cells is similar to that obtained with human lung fibroblasts (not shown). These results show that the expression of ICBP90 is concomitant with the expression of TopoII{alpha}. As a control, Fig. 5CCitation shows the expression of actin (42 kDa) in HeLa cells and in lung fibroblasts, at confluence or in proliferation.



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  		Fig. 5. Expression of ICBP90 (A), TopoII{alpha} (B), and actin (C) in HeLa cells and human lung fibroblasts analyzed by Western blot. Total proteins from cell lysates (10 µg) from nongrowing, confluent HeLa and human fibroblast cells (Lanes 1 and 3, respectively) and from proliferating HeLa cells and human lung fibroblasts (Lanes 2 and 4, respectively) were electrophoresed in SDS-8% polyacrylamide gel and transferred onto nitrocellulose membrane.

 
ICBP90 Is a CCAAT Binding Protein That Contributes to the Expression of TopoII{alpha}.
The effect of the transfection of COS-1 cells with pSG5 containing the cDNA of ICBP90 on the expression of the endogenous TopoII{alpha} is illustrated in Fig. 6ACitation . In nontransfected COS-1 cells, i.e., incubated 24 h with FuGENE only, ICBP90 was detected at 97 kDa (Lane 1), and TopoII{alpha} was detected at 170 kDa (Lane 3). In COS-1-transfected cells, an enhanced expression of ICBP90 (Lane 2) and TopoII{alpha} (Lane 4) were detected. The enhanced expression of ICBP90 is accompanied by the appearance of several bands, as was observed for proliferating HeLa cells. The increase in TopoII{alpha} expression is also accompanied by an increase of other bands, which probably results from an increase in protein catabolism. Fig. 6BCitation shows the electrophoretic migration shift assays performed with 1 µg of ICBP90 per well. Using 32P-end-labeled oligonucleotides containing the ICB2, ICBP90 was shown to be able to bind to ICB2 (Lane 2). The binding of ICBP90 to the labeled probe was partially competed by 0.5 ng (Lane 3) and completely competed by 2.5 ng (Lane 4) and 5 ng (Lane 5) of nonlabeled ICB2. In contrast, an oligonucleotide containing a mutated ICB (ICB2m) was unable to significantly compete the binding at 2.5 ng (Lane 6) or at 5 ng (Lane 7). This demonstrates that ICBP90 binds to ICB2 in a specific manner.



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  		Fig. 6. Binding of ICBP90 to ICB2 and the effect of the overexpression of ICBP90 on the expression of the endogenous COS-1 TopoII{alpha}. Expression of ICBP90 and TopoII{alpha} in COS-1 cells were analyzed by Western blot (A). Nontransfected COS-1 cells (Lanes 1 and 3) were incubated 24 h with FuGENE (30 µl), whereas transfected COS-1 cells (Lanes 2 and 4) were incubated 24 h with 30 µl of FuGENE and 10 µg of pSG5 containing the cDNA of ICBP90. Total proteins from cell lysates (10 µg) were electrophoresed in SDS-8% polyacrylamide gel and transferred onto a nitrocellulose membrane. Binding of ICBP90 to ICB2 was analyzed by electrophoretic migration shift assay (B). Labeled ICB2 was added at 0.5 ng/well, and 1x, 5x, and 10x of nonlabeled ICB2 correspond to 0.5, 2.5, and 5 ng/well, respectively; 5x and 10x of nonlabeled ICB2m correspond to 2.5 and 5 ng/well, respectively. Control, C, was performed in the absence of added proteins. Assays and sequences of nucleotides are described in "Materials and Methods." The positions of bound, B, and free, F, probes are indicated.

 
Expression of ICBP90 and TopoII{alpha} in Tissues from Human Appendix and Primary Breast Cancer.
Fig. 7Citation shows the expression of ICBP90 and TopoII{alpha} in paraffin-embedded human appendix tissues and primary breast carcinomas. In the appendix tissue, it is well known that the proliferative areas are restricted to the germinal centers and the glandular crypts. The immunostaining with an anti-TopoII{alpha} mAb shows that cells localized in the glandular crypts are positive (Fig. 7A)Citation . When using the 1RC1C-10 mAb (anti-ICBP90 mAb), the labeling was also limited to the glandular crypts (Fig. 7B)Citation .



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  		Fig. 7. Immunoperoxidase staining on serial adjacent sections separated by 3 µm, of the nucleus of cells in proliferating area (GC, glandular crypts), of the human appendix (A and B), and in breast carcinoma (C and D) with anti-TopoII{alpha} (A and C) and anti-ICBP90 (B and D) antibodies. The 1RC1C-10 mAb (0.2 µg/ml) and the anti-TopoII{alpha} mAb (0.2 µg/ml) were incubated overnight at room temperature. Mitotic cells are indicated by an arrow. Original magnification: x400.

 
The expression of ICBP90 and TopoII{alpha} was investigated in high-grade or low-grade primary breast carcinomas. The expression of TopoII{alpha} is detectable in several cells with a variable intensity (Fig. 7C)Citation . These cells can be considered as proliferating cells because TopoII{alpha} is known to be expressed in the S phase until the M phase with maximal expression during G2-M (11 , 12) . We found in high-grade breast carcinomas 23.4% and 13.3% of positive cells for ICBP90 and TopoII{alpha}, respectively, whereas in low-grade breast carcinomas, 6.9% and 2.8% of the cells were positive for ICBP90 and TopoII{alpha}, respectively (data not illustrated). In mitotic cells (see arrows in Fig. 7Citation ), the expression of TopoII{alpha} appears to be higher than that of ICBP90. However, it is possible that the disruption of the nuclear membrane led to the dilution of ICBP90 into the cytoplasm, whereas TopoII{alpha} remained on the chromosomes.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have isolated a cDNA coding for a novel human protein that we called ICBP90, because it is an Inverted CCAAT box Binding Protein with a calculated size of 89,758 Da. Immunostaining experiments demonstrate that endogenous and overexpressed ICBP90 are located in the nucleus of HeLa and COS-1 cells, respectively. This is consistent with the presence of two potential nuclear localization signals found in the deduced ICBP90 amino acid sequence. No significant homology, except for the PHD finger (Fig. 3)Citation and the ubiquitin-like domain, was found with other proteins, some of which are known to be transcription factors. This suggests that ICBP90 may be a member of a new family of nuclear proteins with DNA-binding properties. Consistent with such an idea, recently, a mouse nuclear protein of 781 amino acids (Np95) was sequenced (39) . Np95 shows a 73.7% identity with ICBP90, which is not sufficient to conclude that Np95 is the mouse counterpart of ICBP90. Indeed, differences exist between these two proteins in terms of amino acid sequences, as well as in tissue distribution. For example, in the Np95 protein, an ATP/GTP-binding site motif between A296 and S303 (ALRNTGKS) and an A/E-cdk2 binding site were described, whereas we did not find such motifs in ICBP90.

Zinc finger domains are structural motifs for DNA recognition (40) . A zinc finger of the PHD finger type was found in ICBP90 between N310 and D366. A PHD finger is a C4HC3 zinc-finger motif found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation (41) . This suggests that the zinc-finger motif is the DNA-binding domain of ICBP90. The ring finger between C724 and R763 could be involved in the interaction with other proteins. Interestingly, the ring finger was found to be homologous to the zinc finger of RAG1, a mouse transcription factor; in this case, the zinc finger is involved in the dimerization process (42) . Therefore, it is possible that the active form of ICBP90 may be a dimer.

We examined the expression pattern of ICBP90 and TopoII{alpha} in human lung fibroblasts (nontumoral), HeLa cells (tumoral), and COS-1 transfected cells. ICBP90 and TopoII{alpha} were found to be expressed in human lung fibroblasts only when they were proliferating. In HeLa cells, ICBP90 as well as TopoII{alpha} were found in proliferating cells but also in confluent cells with further serum restriction. This can be explained by the fact that in HeLa cells, TopoII{alpha} is detected throughout the cell cycle with a 3-fold increase during S and G2 phases (14) . Because HeLa cells are unable to leave the cell cycle, i. e., enter into the G0 phase, even at confluence, it was not surprising to find TopoII{alpha} and ICBP90 in HeLa cells at confluence. At least confluence diminished the expression of TopoII{alpha} and prevented the appearance of additional bands of ICBP90 when compared to proliferating HeLa cells. It has been reported that the expression of TopoII{alpha} was sensitive neither to serum restriction nor to cell density inhibition, whereas human skin fibroblasts were (43) , as shown in the present study. HeLa cells or MOLT-4, HL60, and Jurkat cells appear to express more ICBP90 than growing fibroblasts or human bronchial smooth muscle cells (data not shown), suggesting that in tumoral cells, there is an enhanced expression of ICBP90. The mechanism of this deregulation is not yet elucidated but might involve gene activation, gene mutations, or alteration of promoter activity, known mechanisms in carcinogenesis (44) . Considering that programmed degradation of many cell cycle regulators, such as cyclins, are key mechanisms in cell-cycle regulation (45) , we suppose that during proliferation of HeLa cells, the appearance of the other bands (<97 kDa) might be related to enhanced ICBP90 catabolism. In this way, the ubiquitin-like domain of ICBP90 may play an important role in this regulating mechanism. Consistent with this hypothesis, the overexpression of ICBP90 in transfected COS-1 cells led to the appearance of several bands, although different from those seen in HeLa cells. The overexpression of ICBP90 led to an interesting result; namely, we observed that 24 h after the transfection, there was an enhanced expression of TopoII{alpha}. This result suggests that ICBP90 participates in the cellular mechanisms controlling the expression of TopoII{alpha}. In HeLa cells and to a lesser extent in human lung fibroblasts, a slight band >97 kDa was observed (Fig. 5)Citation . The significance of the doubling of the band remains unresolved, but it might result from different posttranslational regulation processes, e.g., phosphorylation. This process is of particular importance for transcription factors (46 , 47) .

The involvement of ICBP90 in cell proliferation is supported by four arguments. First, ICBP90 does not appear to be expressed in highly differentiated tissues such as the central nervous system. The ICBP90 mRNA is most abundant in thymus, fetal thymus, fetal liver, and bone marrow. This is possibly linked to their proliferating status but other tissues undergoing cell proliferation, for example, spleen and fetal brain showed weak ICBP90 mRNA, suggesting an additional tissue specificity. Interestingly, peripheral leukocytes do not express ICBP90. Consistent with this, the expression of TopoII{alpha} was found to be the highest in human bone marrow-enriched progranulocytes and myelocytes and decreased during maturation (48) . The second argument is the different pattern of ICBP90 expression in proliferating cells, which is probably linked to enhanced metabolism in tumoral cells and to an increase in the transcription in nontumoral cells. The third argument lies in the observation that the mAb 1RC1C-10 solely labels the nucleus of cells in the proliferating area in paraffin-embedded human appendix tissues (base of glandular crypts). Finally, the observation that ICBP90 colocalizes with TopoII{alpha} in high-grade breast cancer tissue is in agreement with a possible involvement of ICBP90 in cell proliferation.

Because ICBP90 is a DNA-binding protein recognizing a CCAAT box, and CCAAT boxes are important in the promoter of the TopoII{alpha} gene (21 , 28 , 29 , 49) , it is reasonable to propose that beside NF-Y (28) , ICBP90 participates in the regulation of TopoII{alpha} expression. Consistent with such an idea, two unidentified proteins with estimated sizes of 90 and 140 kDa were shown to bind to the ICBs of the TopoII{alpha} gene promoter (29) . Therefore, it would be tempting to propose that this 90 kDa may be ICBP90, but this requires further investigations. Several other promoters have CCAAT boxes in the inverted position or not, and therefore, it is possible that ICBP90 acts on the expression of other genes involved in cell proliferation, with subsequent effects on the expression of TopoII{alpha}.

In summary, the present data show that ICBP90 may regulate TopoII{alpha} expression through an activation of the TopoII{alpha} gene promoter. Also, ICBP90 seems to be strongly involved in cell proliferation processes and in cancer mechanisms, which make it highly interesting for cancer diagnosis, prognosis, and therapy. If ICBP90 and Np95 effectively belong to the same putative family of proteins involved in cell proliferation, it is interesting to mention that Np95 is up-regulated in the S phase and down-regulated in G2-M in normal mouse T-cells, whereas in the tumor T-cell, its expression appears to be constant throughout the cell cycle (50) .


   ACKNOWLEDGMENTS
 
Professor Pierre Chambon is acknowledged for his constructive and continuous support. We thank Danielle Stephan and Serge Vicaire for DNA sequencing, the group of Adrien Staub for oligonucleotide synthesis, and Mustapha Oulad for his technical support. We also thank Sydney Shall for critical reading of the manuscript and Betty Heyd for technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, CNRS, the Hôpital Universitaire de Strasbourg, the Ligue Nationale contre le Cancer, the Comités Départementaux du Bas-Rhin et du Haut-Rhin de La Ligue Nationale contre le Cancer, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, and a fellowship from the Ministère de l’Education Nationale, de la Recherche et de la Technologie (to R. H.). Back

2 To whom requests for reprints should be addressed, at Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, B. P. 163, 67404 Illkirch Cedex, France. Phone: 33-388-65-53-03; Fax: 33-388-65-53-05; E-mail: bronner{at}wotan.u-strasbg.fr Back

3 The abbreviations used are: TopoII, topoisomerase II; ICB, inverted CCAAT box; mAb, monoclonal antibody. Back

4 The amino acid sequence reported in this paper and the corresponding nucleotide sequence have been deposited in the GenBank (accession no. AF129507). All sequences of primers are available on request. Back

Received 6/ 7/99. Accepted 10/28/99.


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 RESULTS
 DISCUSSION
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