Pilot study of Biomarkers for predicting effectiveness of ramosetron in diarrhea-predominant irritable bowel syndrome: expression of S100A10 and polymorphisms of TPH1.

BACKGROUND: Serotonin type 3 receptor (5-HT3 R) antagonists are potentially useful therapeutic agents for diarrhea-predominant irritable bowel syndrome (IBS-D). To identify biomarkers predicting effectiveness of the 5-HT3 R antagonist (ramosetron) in IBS-D. METHODS: Irritable bowel syndrome-D Japanese subjects received 2.5 or 5 µg of ramosetron once daily for 4 weeks. Colonic mucosal S100A and tryptophan hydroxylase (TPH) mRNA expression levels were measured before treatment. Genomic DNA was extracted from blood and polymorphisms of TPH1 and TPH2 were analyzed. KEY RESULTS: Forty-two patients (27 men and 15 women, mean age 42 years) with IBS-D were included for analysis. Improvement of IBS symptoms was seen in 26 (61.9%). Baseline S100A10 (p = 0.02) and TPH1 (p = 0.02) expression were significantly higher in the ramosetron responders than in the non-responders. The frequencies of the TPH1 rs4537731G allele in linkage disequilibrium with the TPH1 rs7130929 T allele (11.5% vs 50%, p = 0.003; OR: 12; 95% CI: 2.1-69) along with TPH1 rs211105 C allele (3.8% vs 43.8%, p = 0.0003; OR: 19; 95% CI: 2.1-181) were significantly lower in the responders than in the non-responders. The mean scores of diarrhea at baseline were significantly higher (5.2 vs 3.7, p = 0.005) in patients with TPH1 rs211105 T/T than those with the G allele. CONCLUSIONS & INFERENCES: TPH1 gene polymorphisms and S100A10 expression, which correlate with 5-HT signaling were associated with ramosetron effectiveness in IBS-D, and may possibly lead to prospective identification of the resistance to treatment.

Tissue stem cells and cancer stem cells: potential implications for gastric cancer.

Gastric cancer remains the second leading cause of death in the world today, making the search for its molecular and cellular basis an important priority. Though recognition of the tight link between inflammation and tumorigenesis is centuries old, only recently are the pieces of the etiological puzzle beginning to fall together. Recent advances in gastric stem cell biology appear to be central to this slowly resolving puzzle. At least two types of stem cells may be important. Resident adult or tissue stem cells may, in a chronically inflamed environment, slowly acquire a series of genetic and epigenetic changes that lead to their emergence as ''cancer stem cells''. This scenario has not yet been proven experimentally, although the first step, prospective recognition of a gastric stem cell has recently been conquered. Alternatively, the setting of chronic inflammatory stress and injury may lead to loss of the indigenous gastric stem cells from their niches; bone marrow derived stem cells may then be recruited to and engraft into the gastric epithelium. Such recruited cells have the potential to contribute to the tumor mass. Indeed, evidence supporting this scenario has been published. Here, we review these recent findings and discuss implications for the future.

Helicobacter pylori-induced atrophic gastritis progressing to gastric cancer exhibits sonic hedgehog loss and aberrant CDX2 expression.

BACKGROUND: The loss of sonic hedgehog is an early change that occurs in the mucosa prior to neoplastic transformation and correlates with the type of intestinal metaplasia. Aberrant expression of CDX has also been shown to correlate with the development of intestinal metaplasia. AIM: To examine CDX2 expression in the non-cancerous mucosa of patients with gastric cancer and compared it to CDX2 expression in controls with intestinal metaplasia. METHODS: Sixty patients who had undergone endoscopic mucosal resection for early gastric cancer and 60 gender- and age-matched controls were studied. Two specimens each were obtained from the greater and lesser curves of the corpus and from the greater curve of the antrum. Expression of CDX2 and sonic hedgehog were evaluated by immunostaining. RESULTS: Gastric cancer was associated with a higher frequency of incomplete intestinal metaplasia (OR = 8.3; 95%CI, 3.7-18.9, P < 0.001). CDX2 negatively correlated with sonic hedgehog expression, however, multivariate analysis revealed that CDX2 correlated with the intestinal metaplasia scores. Sonic hedgehog indices were lower and CDX2 staining in the corpus lesser curve was higher in the cancer group than in the controls. Sonic hedgehog indices in the corpus decreased and CDX2 indices in both areas increased in patients in the ascending order of those without intestinal metaplasia, those with complete intestinal metaplasia and those with incomplete intestinal metaplasia (P < 0.001). CONCLUSIONS: Loss of sonic hedgehog expression and aberrant expression of CDX2 correlates with the type of intestinal metaplasia and may play a role in carcinogenesis.

ZBP-89-induced apoptosis is p53-independent and requires JNK.

ZBP-89 induces apoptosis in human gastrointestinal cancer cells through a p53-independent mechanism. To understand the apoptotic pathway regulated by ZBP-89, we identified downstream signal transduction targets. Ectopic expression of ZBP-89 induced apoptosis through the mitochondrial pathway and was accompanied by activation of all three MAP kinase subfamilies: JNK1/2, ERK1/2 and p38 MAP kinase. ZBP-89-induced apoptosis was markedly enhanced by ERK inhibition with U0126. In contrast, inhibiting JNK with a JNK1-specific peptide inhibitor or dominant-negative JNK2 expression abrogated ZBP-89-mediated apoptosis. The p38 inhibitor SB202190 had no effect on ZBP-89-induced cell death. Protein dephosphorylation assays revealed that ZBP-89 activates JNK via repression of JNK dephosphorylation. Oligonucleotide microarray analyses revealed that ectopic expression of ZBP-89 downregulated expression of the dual-specificity phosphatase MKP6. Overexpression of MKP6 blocked ZBP-89-induced JNK phosphorylation and PARP cleavage. In addition, ectopic expression of ZBP-89 repressed Bcl-xL and Mcl-1 expression, but had no effect on Bcl-2. Silencing ZBP-89 with small interfering RNA enhanced both Bcl-xL and Mcl-1 expression. Taken together, ZBP-89-mediated apoptosis occurs via a p53-independent mechanism that requires JNK activation.

Gastritis and hypergastrinemia due to Acinetobacter lwoffii in mice.

In mouse models and humans, Helicobacter pylori is associated with an increase in serum gastrin and gastrin-expressing (G) cells with a concomitant decrease in somatostatin-expressing D cells. Inflammation of the gastric mucosa can progress to metaplastic changes in the stomach and to decreased colonization by H. pylori and increased colonization by non-H. pylori organisms. In addition, about 20% of individuals with chronic gastritis are H. pylori negative, suggesting that other organisms may induce gastritis. Consistent with this hypothesis, we report here that Acinetobacter lwoffii causes the same histologic changes as does H. pylori. Gastric epithelial cells were isolated from the entire stomach by an enzymatic method for quantitation by both flow cytometry and morphometric analysis. Two months after mice were inoculated with H. pylori or A. lwoffii, the mucosal T- and B-cell numbers significantly increased. After 4 months of infection, there was a threefold increase in the number of G cells and a doubling in the number of parietal cells. A threefold decrease in the number of D cells occurred in H. pylori- and A. lwoffii-infected mice. Plasma gastrin levels increased after both H. pylori and A. lwoffii infection. Histology revealed the presence of inflammation in the gastric mucosa with both A. lwoffii and H. pylori infection. A periodic acid-Schiff stain-alcian blue stain revealed mucous gland metaplasia of the corpus. Collectively, the results demonstrate that gastritis and hypergastrinemia are not specific for H. pylori but can be induced by other gram-negative bacteria capable of infecting the mouse stomach.

TNF-alpha and interleukin 1 activate gastrin gene expression via MAPK- and PKC-dependent mechanisms.

Helicobacter pylori and proinflammatory cytokines have a direct stimulatory effect on gastrin release from isolated G cells, but little is known about the mechanism by which these factors regulate gastrin gene expression. We explored whether tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 directly regulate gastrin gene expression and, if so, by what mechanism. TNF-alpha and IL-1 significantly increased gastrin mRNA in canine G cells to 181 +/- 18% and 187 +/- 28% of control, respectively, after 24 h of treatment. TNF-alpha and IL-1 stimulated gastrin promoter activity to a maximal level of 285 +/- 12% and 415 +/- 26% of control. PD-98059 (a mitogen-activated protein kinase kinase inhibitor), SB-202190 (a p38 kinase inhibitor), and GF-109203 (a protein kinase C inhibitor) inhibited the stimulatory action of both cytokines on the gastrin promoter. In conclusion, both cytokines can directly regulate gastrin gene expression via a mitogen-activated protein kinase- and protein kinase C-dependent mechanism. These data suggest that TNF-alpha and IL-1 may play a direct role in Helicobacter pylori-induced hypergastrinemia.

ZBP-89, Sp1, and nuclear factor-kappa B regulate epithelial neutrophil-activating peptide-78 gene expression in Caco-2 human colonic epithelial cells.

We reported previously that human colonic epithelial cells produce the C-X-C chemokine epithelial neutrophil-activating peptide-78 (ENA-78) and that its expression is up-regulated in ulcerative colitis. The aim of this study was to investigate the transcriptional regulation of ENA-78 gene expression in Caco-2 intestinal epithelial cells. Reporter gene transfection and electrophoretic mobility shift assay studies demonstrated that cooperation between two regions of the ENA-78 promoter were required for maximal gene expression in interleukin-1beta-stimulated Caco-2 cells. Binding of activated p50/p65 nuclear factor-kappaB to nucleotides -82 to -91 was essential for interleukin-1beta-dependent gene transcription, whereas binding of constitutively expressed zinc-requiring nuclear factors to nucleotides -125 to -134 (site A) was required for basal gene expression. Scanning mutagenesis of site A demonstrated overlapping binding elements at this locus. One site (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) up-regulated basal ENA-78 transcription. Another site (CCCCTCCCCC) was found to bind the zinc finger nuclear factor ZBP-89, and overexpression of this protein significantly repressed ENA-78 reporter gene activity. This study demonstrates that ENA-78 gene expression in Caco-2 intestinal epithelial cells is subject to complex regulation involving the coordinate binding of ZBP-89, Sp1, and nuclear factor-kappaB to the ENA-78 promoter.

ZBP-89 promotes growth arrest through stabilization of p53.

Transcription factor p53 can induce growth arrest and/or apoptosis in cells through activation or repression of downstream target genes. Recently, we reported that ZBP-89 cooperates with histone acetyltransferase coactivator p300 in the regulation of p21(waf1), a cyclin-dependent kinase inhibitor whose associated gene is a target gene of p53. Therefore, we examined whether ZBP-89 might also inhibit cell growth by activating p53. In the present study, we demonstrate that elevated levels of ZBP-89 induce growth arrest and apoptosis in human gastrointestinal cell lines. The ZBP-89 protein accumulated within 4 h, and the p53 protein accumulated within 16 h, of serum starvation without changes in p14ARF levels, demonstrating a physiological increase in the cellular levels of these two proteins. Overexpression of ZBP-89 stabilized the p53 protein and enhanced its transcriptional activity through direct protein-protein interactions. The DNA binding and C-terminal domains of p53 and the zinc finger domain of ZBP-89 mediated the interaction. A point mutation in the p53 DNA binding domain, R273H, greatly reduced ZBP-89-mediated stabilization but not their physical interaction. Furthermore, ZBP-89 formed a complex with p53 and MDM2 and therefore did not prevent the MDM2-p53 interaction. However, heterokaryon assays demonstrated that ZBP-89 retained p53 in the nucleus. Collectively, these data indicate that ZBP-89 regulates cell proliferation in part through its ability to directly bind the p53 protein and retard its nuclear export. Our findings further our understanding of how ZBP-89 modulates cell proliferation and reveals a novel mechanism by which the p53 protein is stabilized.

Retinoic acid (RA) receptor transcriptional activation correlates with inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase (ODC) activity by retinoids: a potential role for trans-RA-induced ZBP-89 in ODC inhibition.

Evaluation of retinoic acid receptor (RAR) subtype-selective alpha and gamma agonists and antagonists and a retinoid X receptor (RXR) class-selective agonist for efficacy at inhibiting both induction of ornithine decarboxylase (ODC) by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis and rat tracheal epithelial cells and the appearance of papillomas in mouse epidermis treated in the 2-stage tumor initiation-promotion model indicated that (i) RXR class-selective transcriptional agonists, such as MM11246, were not involved in ODC inhibition; (ii) RAR-selective agonists that induce gene transcription from RA-responsive elements (RAREs) were active at low concentrations; (iii) RAR-selective antagonists that bind RARs and inhibit AP-1 activation on the collagenase promoter but do not activate RAREs to induce gene transcription were less effective inhibitors; and (iv) RARgamma-selective retinoid agonists were more effective inhibitors of TPA-induced ODC activity than RARalpha-selective agonists. These results suggest that RARE activation has a more important role in inhibition of ODC activity than RXR activation or AP-1 inhibition and that RARgamma-selective agonists would be the most useful inhibitors of epithelial cell proliferation induced by tumor promoters. The natural retinoid all-trans-RA induced expression of transcription factor ZBP-89, which represses activation of the GC box in the ODC promoter by the transcription factor Sp1.

EGF receptor activation of the human gastrin gene: a tale of two zinc finger transcription factor families.

Gastrin is one of the oldest and most actively studied of the gastrointestinal peptides. Adult expression of the mature peptide is restricted to the stomach and duodenum. However, it is antral gastrin that is subject to feedback regulation from the counter-regulatory peptide somatostatin as a function of gastric pH. Yet little is known about the molecular steps required to modulate the synthesis of this peptide. Using cell culture models, a GC-rich regulatory element called gERE has been identified that binds two families of zinc finger transcription factors--Sp and ZBP. Competitive binding of these two transcription factor families in association with changes in their phosphorylation state appears to mediate the positive and negative activation of this gene in response to EGF receptor ligands. These findings contribute to our understanding of gastrin gene expression in the presence of the activated ras oncogene, e.g., during colonic transformation.

Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.

Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(waf1) transcription is mediated through proximal GC-rich sites. Prior studies have shown that Sp1, Sp3, and the histone acetylase co-activator p300 are components of the complexes binding to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. This study sought to determine whether ZBP-89 rather than Sp1 is the direct target of p300 during butyrate induction of p21(waf1). ZBP-89 (BFCOL1, BERF-1, ZNF 148) is a Krüppel-type zinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. Adenoviral-mediated expression of ZBP-89 in HT-29 cells revealed that ZBP-89 potentiates butyrate induction of endogenous p21(waf1) gene expression. Further, cotransfection of a ZBP-89 expression vector with a 2.3-kilobase p21(waf1) reporter recapitulated the potentiation by butyrate. DNase I footprinting analysis of the human p21(waf1) promoter with recombinant ZBP-89 identified a binding site at -245 to -215. Electrophoretic mobility shift assays confirmed that both recombinant and endogenous ZBP-89 and Sp1 bind to this element. The potentiation was abolished in the presence of adenoviral protein E1A. Deletion of the N-terminal domain of ZBP-89 abolished the potentiation mediated by butyrate treatment. This same deletion mutant abolished the ZBP-89 interaction with p300. Cotransfection of p300 with ZBP-89 stimulated the p21(waf1) promoter in the absence of butyrate. p300 co-precipitated with ZBP-89 but not with Sp1, whereas ZBP-89 co-precipitated with Sp1. Together, these findings demonstrate that ZBP-89 also plays a critical role in butyrate activation of the p21(waf1) promoter and reveals preferential cooperation of this four-zinc finger transcription factor with p300.

Use of flow cytometry to quantify mouse gastric epithelial cell populations.

Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension. The quality of flow cytometry is strongly dependent on the isolation of intact viable cells. However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated. The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry. Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation. A Percoll density gradient was used to separate viable cells from cellular debris. Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry. According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10-20%) compared to enzymatic separation (1%). After Percoll centrifugation there was a further increase in the percent of viable cells (50-80%). Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively. T and B lymphocytes made up 4% and 2% in the gastric mucosa. Dissociated splenocytes were comprised of 20% T cells and 14% B cells. The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods. Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.

An OmpA-like protein from Acinetobacter spp. stimulates gastrin and interleukin-8 promoters.

Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 10(8) organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified an Acinetobacter sp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of the Acinetobacter sp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach.

The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1.

Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.

EGF stimulates gastrin promoter through activation of Sp1 kinase activity.

Epidermal growth factor (EGF) receptor activation stimulates gastrin gene expression through a GC-rich element called gastrin EGF response element (gERE). This element is bound by Sp1 family members and is a target of the ras-extracellular signal-regulated kinase (Erk) signal transduction cascade. This raised the possibility that Sp1 may be phosphorylated by kinases of this signaling pathway. Erk is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk and Sap, suggesting that Erk may also mediate EGF-dependent phosphorylation of Sp1. This possibility was tested by studying Sp1-dependent kinase activity in extracts prepared from EGF-activated AGS cells by use of solid-phase kinase assays and immunoprecipitation of metabolically labeled Sp1. The results revealed that Sp1 kinase activity (like gastrin promoter activation) is inhibited by PD-98059 and, therefore, is dependent on mitogen-activated protein kinase kinase 1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not account for most of the Sp1 kinase activity, since Erk and additional Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from an ion-exchange column in different fractions. Phosphoamino acid analysis of in vivo radiolabeled Sp1 demonstrated that the kinase phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk.

ZBP-99 defines a conserved family of transcription factors and regulates ornithine decarboxylase gene expression.

Among transcription factors that regulate ornithine decarboxylase (ODC) gene expression are those that interact with GC-rich promoters, including Sp1 and ZBP-89. Sp1 functions as a transactivator and ZBP-89 as a transrepressor of both the ODC and gastrin promoters. This study reports the cloning and characterization of a second member of the ZBP family that also binds GC boxes. ZBP-99 contains four Krüppel-type zinc fingers that collectively share 91% amino acid sequence similarity and 79% sequence identity with those found in ZBP-89. In addition, there are highly conserved amino acid sequences in the carboxy-terminal segments of the two genes. In spite of their structural similarities, the two proteins are encoded at distinct loci, ZBP-89 on chromosome 3q21 and ZBP-99 on 1q32.1. The predicted open reading frame of ZBP-99 cDNA encodes a 99-kDa protein. Electrophoretic mobility shift assays showed that ZBP-99 protein specifically binds to the GC-rich promoter elements of gastrin and ODC genes. Northern blot analysis showed that a major ZBP-99 transcript of 5.6 kb is expressed ubiquitously at low levels, with elevated expression levels in placenta and in adult kidney, liver, and lymphocytes. Cotransfection of AGS gastric adenocarcinoma and HT-29 colon adenocarcinoma cells with a ZBP-99 expression construct and with an ODC reporter construct show that ZBP-99 repressed basal expression in the two cell lines by 80 and 60%, respectively. Collectively, the data suggest that ZBP-99 binds GC-rich promoters and may complement the activities mediated by ZBP-89.

Sp1 phosphorylation by Erk 2 stimulates DNA binding.

EGF stimulates gene expression through a variety of signal transduction pathways that include the ras-Erk pathway. We have shown previously that EGF receptor activation stimulates gastrin gene expression through a GC-rich element called gERE. This element binds Sp1 family members and raises the possibility that the ras-Erk signal transduction cascade may target this novel EGF responsive element. Moreover, it is known that Erk 2 is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk, Sap suggesting that Erk may also inducibly phosphorylate Sp1. To test this hypothesis directly using cotransfection experiments, we show that ras and Erk 2 activation indeed target the gERE element. The Mek 1 kinase inhibitor, PD98059, blocks 50% of EGF-inducible gastrin promoter activity. Pretreatment of the extracts with recombinant Erk2 stimulated Sp1 binding; whereas dephosphorylation reduced but did not eliminate Sp1 binding. Together, these studies demonstrate the novel finding that inducible binding of Sp1 is regulated by its state of phosphorylation. Further, gastrin promoter activation is mediated in part by the ras-Erk signaling cascade that targets Sp1.

Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.

Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, and in vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider's Drosophila line 2, consistent with properties previously attributed to NF-ODC1.