Gastrin Promoter Research Articles

Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells

Gastrin isoforms, acting through a variety of receptors, have proliferative and anti-apoptotic effects on gastrointestinal (GI) cancers. A small interfering RNA (siRNA) targeting the gastrin gene was used to investigate the role of endogenous gastrin in GI cancer cell survival. Downregulation of the gastrin gene in siRNA-transfected cells was measured using real-time reverse transcriptase–PCR. The most effective siRNA was tested in a panel of GI cancer cell lines at various concentrations and time points, and the effect on cell survival and apoptosis was measured using methyl thiazoyl tetrazolium (MTT) and caspase 3 activation assays. Gastrin siRNA reduced gene expression by more than 90% in a range of GI cancer cell lines. Downregulation of the gastrin gene was dose-dependent and effective over approximately 1 week in vitro. However, downregulation at the protein level was delayed by 3–4 days. Gastrin siRNA-transfected cells showed up to a 60% reduction in growth and up to a 50% increase in apoptosis compared with control siRNA-transfected cells. The effects were most marked in the cell line with the highest constitutive level of gastrin gene expression (human metastatic colon, C170HM2) and in epidermal growth factor (EGF)-treated cells as the gastrin promoter contains an EGF-response element, gERE. The ability of the siRNAs to reduce survival of these GI cell lines is further evidence of the importance of autocrine and/or intracrine gastrin loops in GI cancer, where expression of the gastrin gene and autonomous gastrin appears widespread.

The gastrin gene promoter is regulated by p73 isoforms in tumor cells

p73, a new p53 family member, is a transcription factor that is increasingly recognized in cancer research as an important player in tumorigenesis as well as in chemotherapeutic drug sensitivity. Despite the substantial structural and functional similarities to p53, accumulating evidence suggests that p53 and p73 may differently regulate their transcriptional targets. In this study, we have investigated the role of p73 in regulation of the gastrin gene promoter. Gastrin is a peptide hormone and an important factor in determining the progression of a number of human malignancies. Our results show that p73 can bind to the gastrin promoter. This leads to transcriptional upregulation of gastrin mRNA. We also found that the levels of gastrin and p73 transcripts correlate in primary gastric tumors. Taken together, our results demonstrate a novel mechanism for regulation of gastrin gene transcription and support a concept that p53 and p73 may have different biological roles in tumors.

Synergistic activation of the murine gastrin promoter by oncogenic Ras and β-catenin involves SMAD recruitment

While Wnt and Ras signaling pathways are activated during progression of colorectal cancers, many of their important downstream targets remain to be elucidated. The gastrin gene encodes for a family of peptide growth factors that are commonly upregulated in colorectal neoplasia. Previously, we showed that the Wnt signaling pathway moderately stimulates the gastrin promoter. To determine whether Ras signaling can cooperate with Wnt signaling in transcriptional regulation of gastrin gene expression, we have analyzed the response of murine gastrin promoter–reporter gene constructs to combinations of oncogenic stimulation in transient transfection assays. We found a strong (25- to 40-fold) synergistic stimulation of the gastrin promoter by the combination of oncogenic β-catenin and K-ras overexpression. Deletion analysis localized the response element to an area between −140 and −110 bp upstream in the murine gastrin promoter. Electrophoretic mobility shift assays detected a complex containing β-catenin/TCF, AP1, and SMAD3/4 transcription factors that bound to a DNA element through AP1 and SMAD binding sites. Gastrin promoter activation could be further enhanced or suppressed by the co-expression of wild type SMAD4 or dominant negative mutant of SMAD4, respectively, and abrogated by the PI3K inhibitor, LY20004, but not by the MEK inhibitor, PD98059. Taken together, our data strongly suggest that oncogenic Wnt and Ras signaling pathways can synergistically induce gastrin expression, possibly contributing to neoplastic progression.

The Epstein-Barr Virus Protein BMRF1 Activates Gastrin Transcription

The Epstein-Barr virus (EBV) BMRF1 gene encodes an early lytic protein that functions not only as the viral DNA polymerase processivity factor but also as a transcriptional activator. BMRF1 has been previously shown to activate transcription of an EBV early promoter, BHLF1, though a GC-rich motif which binds to SP1 and ZBP-89, although the exact mechanism for this effect is not known (D. J. Law, S. A. Tarle, and J. L. Merchant, Mamm. Genome 9:165-167, 1998). Here we demonstrate that BMRF1 activates transcription of the cellular gastrin gene in telomerase-immortalized keratinocytes. Furthermore, BMRF1 activated a reporter gene construct driven by the gastrin promoter in a variety of cell types, and this effect was mediated by two SP1/ZBP-89 binding sites in the gastrin promoter. ZBP-89 has been previously shown to negatively regulate the gastrin promoter. However, ZBP-89 can function as either a negative or positive regulator of transcription, depending upon the promoter and perhaps other, as-yet-unidentified factors. BMRF1 increased the binding of ZBP-89 to the gastrin promoter, and a ZBP-89-GAL4 fusion protein was converted into a positive transcriptional regulator by cotransfection with BMRF1. BMRF1 also enhanced the transcriptional activity of an SP1-GAL4 fusion protein. These results suggest that BMRF1 activates target promoters through its effect on both the SP1 and ZBP-89 transcription factors. Furthermore, as the EBV genome is present in up to 10% of gastric cancers, and the different forms of gastrin are growth factors for gastrointestinal epithelium, our results suggest a mechanism by which lytic EBV infection could promote the growth of gastric cells.

The Murine Gastrin Promoter Is Synergistically Activated by Transforming Growth Factor-β/Smad and Wnt Signaling Pathways

The transforming growth factor-beta (TGF-beta) and Wnt/wingless pathways play critical roles in the specification of cell fate during development and also contribute to cancer formation and progression. Whereas Wnt signaling is clearly pro-oncogenic, TGF-beta signaling is cell- and context-dependent, manifesting both inhibitory and proliferative effects. The growth factor, gastrin, has previously been shown to be a downstream target of the Wnt pathway and a promoter of gastrointestinal cancer. In this study, we show that the mouse gastrin promoter is regulated synergistically by TGF-beta/Smads and beta-catenin/T-cell factor (TCF). Co-transfection of Smad3/Smad4 and beta-catenin expression constructs synergistically activated mouse gastrin promoter activity 30-60-fold in AGS cells with minimal effect seen with either construct alone. This activation was further potentiated by TGF-beta1 treatment. Mutating either the TCF binding site or the Smad-binding element (SBE) diminished the activation of gastrin expression by Smad3/Smad4 and beta-catenin and led to a loss of gastrin promoter responsiveness to TGF-beta1 treatment. Wnt and TGF-beta regulated endogenous gastrin mRNA levels in AGS cells in a similar fashion, as revealed by small interference RNA studies or overexpression of Smads and TCF4/beta-catenin. Electrophoretic mobility shift assays and DNA affinity precipitation assays showed that the putative SBE and T-cell factor (TCF) sites were able to bind a complex containing Smads and beta-catenin/TCF4. In addition, the synergy between Smads and beta-catenin/TCF4 was dependent on CREB-binding protein (CBP)/P300, as demonstrated by overexpression of CBP or E1A. Moreover, by using a heterogeneous promoter reporter system, we showed that this complex containing Smads/TCF4/beta-catenin complex was able to up-regulate transcription at isolated SBE or TCF sites. Thus, the Wnt signaling pathway is able to activate some target genes through its actions as a co-activator at non-TCF sites and has the potential to profoundly alter transcriptional responses to TGF-beta signaling.

Gastrin expression is regulated by the interplay of TGF-beta/smads and Wnt pathways

Background and Aims: Transtorming growth factor !3 (TGF-13)/gmads and Wnt pathways play a critical role in both repressing and progressing gastrointestinal cancers. Various mutations of TI3RII, Smad2, Smad4, APC, and [3-eatenin are associated with a large nmnber cases of gastric, pancreatic, and colon cancers. Furthermore, gastrin and its precursors (progastrin and Glycine-extended gastrin) have been shown to promote cell proliferation and cancer progression in gastrointestii~al system botb in vitro and in ,Avo. in the effort to integrate the roles and rdationship of TGF-]3 Wm pathway and gastrin in gastric cancer progressmn, we investigated the regulation of gastrin expression by TGF-~/Smads and Wm pa(hways. Methods: Serial deletion and intensive mutational analyses were performed to identit}, the cis<lements regulating mouse gastrin expression. Wild type and mutant mouse gastrin promoter constructs were transiently' transtected into AGS cells with or without transcription tactors. Transfected cells were treated with or without 3ng/mL TGF-[31 for 48 b o u ~ The transcription of mouse gastrin gene was then measured by firefly' lueiferase activity. Results: In AGS gastric adenocarcinoma cell line, overexpression of Smad3/Smad4 and ~-catenm synergistically activated mouse gastrin transcription about 10 to 20 folds while no or minimal activating etlect was detected when Samd3/Smad4 or ]3-catenin presented alone. This aclivation was ff~rther potentiated by TGF-I~I treatment. Mutating either the TCF4 binding site or the distal Smad-binding site diminished the activation of gastrin expression by Smad3/Smad4 and ~<atenin and led to a loss of responsiveness of gastrin promoter to FGF-~ treatment. Conclusions: These results clearly demonstrate that through interactions with Wnt-signal pathway, TGF-~/Smads pathway transcriptionally regulates gastrin expression

Regulation of the gastrin promoter by AP1 at Sp1 DNA elements

Regulation of the gastrin promoter by AP1 at Sp1 DNA elements

Regulation of epithelial cell growth by ZBP-89: potential relevance in pancreatic cancer.

ZBP-89 (ZNF148) is a Zinc finger Binding Protein of 89 kDa that binds GC-rich DNA elements. Originally, it was expression cloned using a DNA element mediating EGF regulation of the gastrin promoter. ZBP-89 functions as both a transcriptional activator and repressor. A variety of extracellular regulators including TGFbeta, retinoic acid and butyrate stimulate ZBP-89 gene expression. Butyrate activation of p21WAF1 is potentiated by ZBP-89 through the recruitment of the co-activator p300, while chronic stimulation by butyrate increases ZBP-89 gene expression correlating with cell differentiation. ZBP-89 stimulates growth arrest and apoptosis through its ability to bind the p21WAF1 promoter or its ability to form protein-protein interactions with p53. ZBP-89 protein is elevated in a variety of gastrointestinal cancers as well as the pancreas. In particular, ZBP-89 is normally expressed in pancreatic islets and ducts and in about 30% of pancreatic adenocarcinomas.

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.

Expression of the human insulin gene in the gastric G cells of transgenic mice.

The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.

Gastrin is a target of the beta-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene occur in most colorectal cancers and lead to activation of beta-catenin. Whereas several downstream targets of beta-catenin have been identified (c-myc, cyclin D1, PPARdelta), the precise functional significance of many of these targets has not been examined directly using genetic approaches. Previous studies have shown that the gene encoding the hormone gastrin is activated during colon cancer progression and the less-processed forms of gastrin are important colonic trophic factors. We show here that the gastrin gene is a downstream target of the beta-catenin/TCF-4 signaling pathway and that cotransfection of a constitutively active beta-catenin expression construct causes a threefold increase in gastrin promoter activity. APC(min-/+) mice overexpressing one of the alternatively processed forms of gastrin, glycine-extended gastrin, show a significant increase in polyp number. Gastrin-deficient APC(min-/+) mice, conversely, showed a marked decrease in polyp number and a significantly decreased polyp proliferation rate. Activation of gastrin by beta-catenin may therefore represent an early event in colorectal tumorigenesis and may contribute significantly toward neoplastic progression. The identification of gastrin as a functionally relevant downstream target of the beta-catenin signaling pathway provides a new target for therapeutic modalities in the treatment of colorectal cancer.

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.

Composite action of three GC/GT boxes in the proximal promoter region is important for gastrin gene transcription

The proximal region of the human gastrin gene promoter contains three GC/GT boxes at positions −140 to −134 bp, −108 to −102 bp and −67 to −61 bp. In this study we have examined the significance of the three elements, and their role in Sp1 and Sp3 mediated gastrin transcription. In AGS cells, mutation of each of the boxes caused a moderate decrease in promoter activity from 33 to 63%, whereas double or triple mutations reduced activity to 3–12%. In Drosophila cells Sp1 activated the promoter, mainly through the distal GC box. Similarly, co-transfection of heterologous promoter constructs revealed that only the distal GC box increased activation by Sp1. The effect of Sp3 was cell-line dependent, since Sp3 inhibited the gastrin promoter activity in AGS cells and caused a synergistic activation of the Sp1 stimulated gastrin promoter in Drosophila cells. Both effects were dependent on the C-terminal DNA binding domain of Sp3. The results indicates that the combined effect of the GC/GT boxes and the ratio between Sp1 and Sp3 are important for gastrin gene expression.

RIN ZF, a Novel Zinc Finger Gene, Encodes Proteins That Bind to the CACC Element of the Gastrin Promoter

Expression of gastrin, a gut hormone and growth factor, has tissue-specific transcriptional regulation and can be induced in some tumors. Previous studies have shown that a CACC cis-regulatory element is important for transcriptional activation in pancreatic insulinoma cells. To identify CACC-binding proteins, a lambda phage cDNA library derived from a rat insulinoma cell line, RIN 38A, was screened by a Southwestern method. A novel member of the Cys2-His2 zinc finger gene family was cloned and designated RIN ZF, having a cDNA sequence of 3.8 kilobases. One full-length and a shorter splice variant were sequenced and had predicted protein masses of 91.6 and 88.7 kDa. Expression of both splice forms were ubiquitous in fetal and adult rat tissues. Recombinant RIN ZF protein exhibited sequence-specific binding to the gastrin CACC element in a gel mobility shift assay. In transient transfections, both splice variants appeared to have only weak activating effects on gastrin-luciferase reporter gene transcription. Furthermore, RIN ZF coexpression with Sp1 appeared to block the strongly activating effects of Sp1 mediated through the CACC element. These findings suggest that a novel set of zinc finger proteins may help regulate gastrin gene expression by interfering with Sp1 transactivation.

RIN ZF , a Novel Zinc Finger Gene, Encodes Proteins That Bind to the CACC Element of the Gastrin Promoter

RIN ZF , a Novel Zinc Finger Gene, Encodes Proteins That Bind to the CACC Element of the Gastrin Promoter

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 thatrasand 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.

Oncogenic ras induces gastrin gene expression in colon cancer

Background & Aims: The expression of gastrin, as a tumor growth factor, is significantly increased in some colon cancers compared with the low levels found in normal mucosa. The aim of this study was to elucidate the transcriptional mechanisms of gastrin induction in colon cancer. Methods: Gastrin messenger (mRNA) levels and K- ras genotype were determined in colon cancer cell lines and surgical specimens. Colon cancer cells were transfected with oncogenic ras expression vectors, and transcriptional activity was assayed with gastrin-luciferase reporter genes. Results: Colon cancer cell lines and tissues with K- ras mutations all had significantly higher gastrin mRNA levels than those that were ras wild type. Treatment of several ras mutant cell lines with PD98059, an inhibitor of mitogen-activated protein kinase kinase, resulted in a decrease in endogenous gastrin mRNA levels. The effects of ras on gastrin expression appeared to be mediated through the gastrin promoter because transfection of oncogenic ras and activated raf expression vectors both induced gastrin-promoter, luciferase-reporter genes. The inductive effects of oncogenic ras could be blocked by the coexpression of dominant negative forms of raf and extracellular regulated kinase. Conclusions: Oncogenic ras induces gastrin gene expression through activation of the Raf-MEK-ERK signal transduction pathway. GASTROENTEROLOGY 1998;115:1144-1153

The ras-Erk kinase signal transduction pathway stimulates the gastrin promoter through the GC-rich gERE element

The ras-Erk kinase signal transduction pathway stimulates the gastrin promoter through the GC-rich gERE element

The human ZBP-89 homolog, located at chromosome 3q21, represses gastrin gene expression.

Rat ZBP-89cDNA encodes an 89-kDa, zinc finger protein thatbinds a GC-rich element in the human gastrin promoter. Thiselement modulates both basal- and epidermal growth factor(EGF)-induction of gastrin gene expression (Merchant et al. 1995).Coexpression of ZBP-89 and gastrin reporter constructs blocksEGF induction and represses basal gastrin gene expression (Mer-chant et al. 1996). Rat ZBP-89similarly binds to and represses theactivity of the ornithine decarboxylase (ODC) promoter, concomi-tantly inhibiting cell proliferation (Remington et al. 1997). ZBP-89shares 99% amino acid sequence identity with two closely relatedmouse homologs, G-rich box-binding protein (GRBBP; Passantinoet al. 1996) and BFCOL1 (Hasegawa and de Crombrugghe 1997).Mutations of the BFCOL1 binding site in the pro a2(I) collagenpromoter inhibit BFCOL1 binding and result in increased pro-moter activity (Hasegawa and de Crombrugghe, 1997). Collec-tively, these data show that rodent ZBP-89-related genes act astranscriptional repressors by binding to specific GC-rich promoterelements, and, at least in some cases, this activity exerts a regu-latory effect on cell proliferation.The human cDNA, htb, shares about 90% DNA sequenceidentity with the open reading frames of rodent ZBP-89homologs.However, two single-nucleotide deletions in htb cDNA, 38 of thezinc finger domain, cause premature stop codons. As a result, htbencodes a 494 rather than a 794-amino acid protein (Merchant etal. 1996; Wang et al. 1993). The truncated, 49-kDa, htb proteinacts as a transcriptional activator, moderately activating T-cellreceptor promoters (Wang et al. 1993). The structural and func-tional comparisons of rodent ZBP-89homologs with htb suggestedto us that the point deletions that distinguish htb may have orig-inated as somatic mutations of wild type human ZBP-89 in theJurkat cells from which the htb cDNA was isolated. To test thishypothesis, we isolated a full-length (∼89 kDa) human ZBP-89cDNA from a normal leukocyte library, compared genomic local-ization of the corresponding gene with that of htb, and determinedits effects on gastrin gene expression. We report that full-lengthZBP-89 maps to Chr 3q21 where the htb cDNA was mapped(Schuler et al. 1996). Furthermore, like its rat homolog, humanZBP-89 functions as a repressor of gastrin gene expression.The human ZBP-89 cDNA clone hZBP-89-16 contains a 2.4-kb insert and was isolated from a SuperScript (Life Technologies)leukocyte library by positive selection (GeneTrapper, Life Tech-nologies) with oligonucleotides from htb. The open reading frameof hZBP-89-16 encodes a 794-amino acid protein, similar to the ratand mouse homologs, and lacks the internal stop codons of htb.A600-bp,EcoRV fragment from the 58-UTR of ZBP-89-16 cDNAwas used to screen a human BAC library (Genome Systems, St.Louis, Mo.), and overlapping clones B469N19, B460C18, andB502J22 were isolated (Fig. 1). A subcloned 1.2-kbEcoRI frag-ment from B469N19 was sequenced and used to develop an STS(374F/794R) for PCR-based screening of the human CEPH YACDNA matrix pools (Research Genetics, Huntsville, Ala.). The re-sulting YAC/BAC contig shows that the human ZBP-89 genemaps to chromosome band 3q21 (144–146 cM on the genetic map)and, more precisely, is within 100 kb of D3S1551. This agrees withour FISH data showing hybridization to 3q21 using BACB469N19 as a probe (data not shown). This localization is also thesame as reported for htb (Schuler et al. 1996), suggesting that twoalternative forms of human ZBP-89-related protein are derivedfrom a single locus. To better assess this possibility, we analyzedthe coding sequences contained in the ZBP-89 BAC clones.The ZBP-89 DNA sequence derived from BAC B469N19 wascompared with those of related genes (Fig. 2). The human se-quence shares 97% amino acid sequence identity with correspond-

ZBP-89, A Krüppel-Type Zinc Finger Protein, Inhibits Cell Proliferation

ZBP-89 is a Krüppel-type zinc finger transcription factor that binds to GC-rich sequences. Overexpression of this factor prevents EGF induction of the gastrin promoter; therefore, we postulated that ZBP-89 may modulate cellular proliferation. To test this hypothesis, ZBP-89 was overexpressed in immortalized (GH4) and malignant (AGS) cell lines. Growth parameters, e.g.,3H-thymidine, BrdU labeling, flow cytometry and ornithine decarboxylase promoter activity were analyzed. The results show that DNA synthesis is inhibited and progression to S phase is blocked in GH4cells. Collectively, these studies demonstrate that ZBP-89 inhibits cellular proliferation at least in part through its ability to bind and repress ornithine deacarboxlyase promoter activity.