Correlation of Biomarker Expression in Colonic Mucosa with Disease Phenotype in Crohn's Disease and Ulcerative Colitis.

BACKGROUND: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are characterized by chronic intestinal inflammation due to immunological, microbial, and environmental factors in genetically predisposed individuals. Advances in the diagnosis, prognosis, and treatment of IBD require the identification of robust biomarkers that can be used for molecular classification of diverse disease presentations. We previously identified five genes, RELA, TNFAIP3 (A20), PIGR, TNF, and IL8, whose mRNA levels in colonic mucosal biopsies could be used in a multivariate analysis to classify patients with CD based on disease behavior and responses to therapy. AIM: We compared expression of these five biomarkers in IBD patients classified as having CD or UC, and in healthy controls. RESULTS: Patients with CD were characterized as having decreased median expression of TNFAIP3, PIGR, and TNF in non-inflamed colonic mucosa as compared to healthy controls. By contrast, UC patients exhibited decreased expression of PIGR and elevated expression of IL8 in colonic mucosa compared to healthy controls. A multivariate analysis combining mRNA levels for all five genes resulted in segregation of individuals based on disease presentation (CD vs. UC) as well as severity, i.e., patients in remission versus those with acute colitis at the time of biopsy. CONCLUSION: We propose that this approach could be used as a model for molecular classification of IBD patients, which could further be enhanced by the inclusion of additional genes that are identified by functional studies, global gene expression analyses, and genome-wide association studies.

Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria.

Antibodies of the secretory IgA (SIgA) class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR). The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus), we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

Lessons from mother: Long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring.

From birth to adulthood, the gut microbiota matures from a simple community dominated by a few major bacterial groups into a highly diverse ecosystem that provides both benefits and challenges to the host. Currently there is great interest in identifying environmental and host factors that shape the development of our gut microbiota. Breast milk is a rich source of maternal antibodies, which provide the first source of adaptive immunity in the newborn's intestinal tract. In this addendum, we summarize our recent data demonstrating that maternal antibodies in breast milk promote long-term intestinal homeostasis in suckling mice by regulating the gut microbiota and host gene expression. We also discuss important unanswered questions, future directions for research in this field, and implications for human health and disease.

Cooperativity among secretory IgA, the polymeric immunoglobulin receptor, and the gut microbiota promotes host-microbial mutualism.

Secretory IgA (SIgA) antibodies in the intestinal tract form the first line of antigen-specific immune defense, preventing access of pathogens as well as commensal microbes to the body proper. SIgA is transported into external secretions by the polymeric immunoglobulin receptor (pIgR). Evidence is reported here that the gut microbiota regulates production of SIgA and pIgR, which act together to regulate the composition and activity of the microbiota. SIgA in the intestinal mucus layer helps to maintain spatial segregation between the microbiota and the epithelial surface without compromising the metabolic activity of the microbes. Products shed by members of the microbial community promote production of SIgA and pIgR by activating pattern recognition receptors on host epithelial and immune cells. Maternal SIgA in breast milk provides protection to newborn mammals until the developing intestinal immune system begins to produce its own SIgA. Disruption of the SIgA-pIgR-microbial triad can increase the risk of infectious, allergic and inflammatory diseases of the intestine.

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression.

Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

Regulation of the mucosal phenotype in dendritic cells by PPARγ: role of tissue microenvironment.

Mucosal DCs play a critical role in tissue homeostasis. Several stimuli can induce a mucosal phenotype; however, molecular pathways that regulate development of mucosal DC function are relatively unknown. This study sought to determine whether PPARγ contributes to the development of the "mucosal" phenotype in mouse DCs. Experiments demonstrated that PPARγ activation in BMDCs induced an immunosuppressive phenotype in which BMDCs had reduced expression of MHC class II and costimulatory molecules, increased IL-10 secretion, and reduced the ability to induce CD4 T cell proliferation. Activation of PPARγ enhanced the ability of BMDC to polarize CD4 T cells toward iTregs and to induce T cell expression of the mucosal homing receptor, CCR9. Activation of PPARγ increased the ability of BMDCs to induce T cell-independent IgA production in B cells. BMDCs from PPARγ(ΔDC) mice displayed enhanced expression of costimulatory molecules, enhanced proinflammatory cytokine production, and decreased IL-10 synthesis. Contrary to the inflammatory BMDC phenotype in vitro, PPARγ(ΔDC) mice showed no change in the frequency or phenotype of mDC in the colon. In contrast, mDCs in the lungs were increased significantly in PPARγ(ΔDC) mice. A modest increase in colitis severity was observed in DSS-treated PPARγ(ΔDC) mice compared with control. These results indicate that PPARγ activation induces a mucosal phenotype in mDCs and that loss of PPARγ promotes an inflammatory phenotype. However, the intestinal microenvironment in vivo can maintain the mucosal DC phenotype of via PPARγ-independent mechanisms.

Multifactorial patterns of gene expression in colonic epithelial cells predict disease phenotypes in experimental colitis.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five "signature" biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohn's disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. METHODS: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)-2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T-cell-mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. RESULTS: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T-cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. CONCLUSIONS: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients.

HIV protease inhibitors alter innate immune response signaling to double-stranded RNA in oral epithelial cells: implications for immune reconstitution inflammatory syndrome?

In this investigation, several HIV protease inhibitors altered the virally associated, double-stranded RNA (dsRNA)-stimulated, innate immune response. Lopinavir, the most potent inducer of interleukin (IL)-8 expression, also inhibited dsRNA-induced monocyte chemotactic protein 1 expression. Further analyses demonstrated that nuclear factor-κB is required for lopinavir's induction of IL-8. These findings demonstrate that protease inhibitors, such as lopinavir, differentially dysregulate innate immune signaling in a manner that could affect immune (reconstitution) inflammatory responses in oral epithelium.

Regulation of the polymeric immunoglobulin receptor in intestinal epithelial cells by Enterobacteriaceae: implications for mucosal homeostasis.

The commensal microbiota of the human colon profoundly impacts host gene expression and mucosal homeostasis. Secretory IgA antibodies, which influence the composition of the intestinal microbiota and provide immunity against pathogens, are transported across intestinal epithelial cells (IEC) by the polymeric immunoglobulin receptor (pIgR). To compare the effects of different colonic bacteria on pIgR expression, the human IEC line HT-29 was stimulated with various species representing the 4 major phyla of colonic bacteria. Only bacteria from the family Enterobacteriaceae (phylum Proteobacteria) induced expression of pIgR and other target genes of bacterial pattern recognition receptors. HT-29 cells responded to purified ligands for Toll-like receptor (TLR)4 but not TLR2. Expression of pIgR and transport of IgA were significantly reduced in colons of mice deficient in the TLR adaptor MyD88, consistent with a role for TLR signaling in the regulation of pIgR by colonic bacteria. Induction of pIgR expression in HT-29 cells required NF-kappaB signaling but not MAPK signaling, in contrast to the requirement for both NF-kappaB and MAPK signaling for induction of pro-inflammatory genes. These results suggest that commensal Enterobacteriaceae may promote intestinal homeostasis by enhancing pIgR expression in IEC.

HIV protease inhibitors block oral epithelial cell DNA synthesis.

OBJECTIVES: Anti-retroviral therapy regimens that include HIV protease inhibitors (PIs) are associated with diverse adverse effects including increased prevalence of oral warts, oral sensorial deficits and gastrointestinal toxicities suggesting that PIs may perturb epithelial cell biology. To test the hypothesis that PIs could affect specific biological processes of oral epithelium, the effects of these agents were evaluated in several oral epithelial cell-lines. DESIGN: Primary and immortalized oral keratinocytes and squamous carcinoma cells of oropharyngeal origin were cultured in the presence of pharmacologically relevant concentrations of PIs. Their affects on cell viability, cytotoxicity and DNA synthesis were assessed by enzymatic assays and incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA. RESULTS: Viability of primary and immortalized oral keratinocytes as well as squamous carcinoma cells of oropharyngeal origin was significantly reduced by select PIs at concentrations found in plasma. Of the seven PIs evaluated, nelfinavir was the most potent with a mean 50% inhibitory concentration [IC(50)] of 4.1 microM. Lopinavir and saquinavir also reduced epithelial cell viability (IC(50) of 10-20 microM). Atazanavir and ritonovir caused minor reductions in viability, while amprenavir and indinavir were not significant inhibitors. The reduced cell viability, as shown by BrdU incorporation assays, was due to inhibition of DNA synthesis rather than cell death due to cytotoxicity. CONCLUSION: Select PIs retard oral epithelial cell proliferation in a drug and dose-dependent manner by blocking DNA synthesis. This could account for some of their adverse effects on oral health.

The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces.

Secretory antibodies of the immunoglobulin A (IgA) class form the first line of antigen-specific immune protection against inhaled, ingested, and sexually transmitted pathogens and antigens at mucosal surfaces. Epithelial transcytosis of polymeric IgA (pIgA) is mediated by the polymeric immunoglobulin receptor (pIgR). At the apical surface, the extracellular ligand-binding region of pIgR, known as secretory component (SC), is cleaved and released in free form or as a component of secretory IgA (SIgA). SC has innate anti-microbial properties, and it protects SIgA from proteolytic degradation. Expression of pIgR is regulated by microbial products through Toll-like receptor signaling and by host factors such as cytokines and hormones. Recent studies of the structure of the extracellular ligand-binding domain of pIgR have revealed mechanisms by which it binds pIgA and other ligands. During transcytosis, pIgA has been shown to neutralize pathogens and antigens within intracellular vesicular compartments. The recent identification of disease-associated polymorphisms in human pIgR near the cleavage site may help to unravel the mystery of how pIgR is cleaved to SC. The identification of novel functions for SC and SIgA has expanded our view of the immunobiology of pIgR, a key component of the mucosal immune system that bridges innate and adaptive immune defense.

Regulation of polymeric immunoglobulin receptor expression by reovirus.

Polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA and IgA-coated immune complexes from the lamina propria across epithelia and into secretions. The effect of reovirus infection on regulation of pIgR expression in the human intestinal epithelial cell line HT-29 was characterized in this report. Both replication-competent and UV-inactivated reovirus at m.o.i. equivalents of 1-100 p.f.u. per cell upregulated pIgR mRNA by 24 h post-infection and intracellular pIgR protein was increased at 48 h following exposure to UV-inactivated virus. Binding of virus to HT-29 cells was required, as pre-incubating virus with specific antiserum, but not non-immune serum, inhibited reovirus-mediated pIgR upregulation. Endosomal acidification leading to uncoating of virus is a required step for pIgR upregulation, as ammonium chloride or bafilomycin A1 pre-treatment inhibited virus-induced pIgR upregulation. Inhibition experiments using the calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal suggested that calpains are involved in reovirus-mediated pIgR upregulation. Upregulation of pIgR following virus infection appears to be an innate immune response against invading pathogens that could help the host clear infection effectively. Signalling induced by microbes and their products may serve to augment pIgR-mediated transcytosis of IgA, linking the innate and acquired immune responses to viruses.

Regulation of the polymeric Ig receptor by signaling through TLRs 3 and 4: linking innate and adaptive immune responses.

IgA Abs help to maintain homeostasis at mucosal surfaces by promoting defense mechanisms that protect against pathogens while suppressing inflammatory responses to commensal organisms and food Ags. The polymeric Ig receptor (pIgR) mediates transport of IgA across mucosal epithelial cells. We hypothesized that signaling through TLRs may up-regulate pIgR expression by intestinal epithelial cells and thus enhance IgA-mediated homeostasis. To test this hypothesis we treated the HT29 human intestinal epithelial cell line with dsRNA, a ligand for TLR3, or LPS, a ligand for TLR4. Both dsRNA and LPS up-regulated levels of pIgR mRNA and cell surface pIgR protein. By contrast, dsRNA but not LPS up-regulated expression of TLR3 and TLR4 mRNA. However, cell surface expression of both TLR3 and TLR4 was enhanced by treatment of HT29 cells with their respective ligands. Transfection of HT29 cells with wild-type and mutated promoter/enhancer plasmids suggested that TLR3 and TLR4 signal primarily through NF-kappaB to enhance transcription of pIgR mRNA. TLR3 signaling resulted in a more pronounced inflammatory response than did TLR4, as evidenced by up-regulation of the transcription factor IFN regulatory factor-1, chemokines IL-8 and RANTES, and the proinflammatory cytokine TNF. Signaling through LPS/TLR4 appears to up-regulate pIgR expression while minimizing proinflammatory responses, a mechanism that could promote IgA-mediated homeostasis in the presence of commensal Gram-negative bacteria.

Long-term exposure of the HT-29 human intestinal epithelial cell line to TNF causes sustained up-regulation of the polymeric Ig receptor and proinflammatory genes through transcriptional and posttranscriptional mechanisms.

Transport of IgA Abs across intestinal epithelial cells into gut secretions is mediated by the polymeric Ig receptor (pIgR). The cytokine TNF plays a central role in initiating and amplifying inflammatory reactions, and is implicated in the pathogenesis of inflammatory bowel diseases. Acute exposure of intestinal epithelial cell lines to TNF has been shown to up-regulate transcription of genes encoding pIgR and a number of proinflammatory factors, but the effects of chronic exposure to TNF have not been studied. We found that exposure of HT-29 human colon carcinoma cells to TNF for up to 20 days reduced the rate of cell proliferation, but did not cause gross morphological changes. Expression of mRNA encoding pIgR and several proinflammatory genes increased acutely, and then diminished but remained elevated above control levels throughout the experiment. Changes in gene expression were paralleled by increased expression of the transcription factors IFN regulatory factor-1 and the RelB subunit of NF-kappaB. HT-29 cells activated the endogenous TNF gene in response to TNF treatment, but the level of TNF production was insufficient to maintain pIgR and proinflammatory gene expression after withdrawal of exogenous TNF. Chronic exposure to TNF caused a marked increase in pIgR mRNA stability and a small but significant decrease in TNF mRNA stability, but no change in the half-lives of IL-8, c-Myc, and GAPDH. In summary, we observed different effects of acute vs chronic exposure to TNF on gene expression, and found evidence for transcriptional and posttranscriptional regulation of expression of the pIgR.

Down-regulation of the polymeric immunoglobulin receptor in non-small cell lung carcinoma: correlation with dysregulated expression of the transcription factors USF and AP2.

The polymeric immunoglobulin receptor (PIGR) mediates transport of IgA and IgM antibodies across mucosal and glandular epithelia. Several studies have utilized immunohistochemistry to demonstrate that PIGR expression varies in different types of lung carcinoma, and is down-regulated during tumor progression. We have previously shown in cultured tumor cell-lines that basal transcription of the PIGR gene is regulated by the transcription factors USF1, USF2 and AP2. To examine the mechanism by which PIGR expression is down-regulated in lung carcinoma, RNA was microdissected from formalin-fixed, paraffin-embedded lung carcinomas (14 adenocarcinomas and 8 squamous cell carcinomas). Levels of PIGR, USF1, USF2 and AP2-alpha mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction and normalized to mRNA for the housekeeping gene GAPDH. PIGR mRNA levels were decreased in adenocarcinomas and squamous cell carcinomas relative to adjacent non-tumor tissue, and were inversely correlated with stage of differentiation. USF1 and USF2 mRNA levels were reduced in adenocarcinomas relative to non-tumor tissue, while AP2-alpha levels were elevated. Multivariate regression analysis demonstrated that reduced USF2 mRNA and increased AP2-alpha mRNA levels were predictive of down-regulated PIGR mRNA expression in the majority of adenocarcinomas and in moderately differentiated squamous cell carcinomas.

A 5'-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRalpha) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRalpha and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRalpha, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to activate the ACE66 enhancer, most likely through activation of factors binding to the AP1 element. These results identify a novel mechanism of transcriptional enhancement involving ligand-independent activity of the VDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.

Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneumoniae into the epithelium.

Streptococcus pneumoniae binds to the ectodomain of the human polymeric Ig receptor (pIgR), also known as secretory component (SC), via a hexapeptide motif in the choline-binding protein SpsA. The SpsA-pIgR interaction mediates adherence and internalization of the human pathogen into epithelial cells. In this study the results of SpsA binding to human, mouse, and chimeric SC strongly supported the human specificity of this unique interaction and suggested that binding sites in the third and fourth Ig-like domain of human SC (D3 and D4, respectively) are involved in SpsA-pIgR complex formation. Binding of SpsA to SC-derived synthetic peptides indicated surface-located potential binding motifs in D3 and D4. Adherence and uptake of pneumococci or SpsA-coated latex beads depended on the SpsA hexapeptide motif as well as SpsA-binding sites in D3 and D4 of human pIgR. The involvement of D3 and D4 in adherence and invasion was demonstrated by the lack of binding of SpsA-coated latex beads to transfected epithelial cells expressing mutated pIgR. Finally, blocking experiments with chimeric human-mouse SC as well as synthetic peptides indicated the participation of D3 and a key role of D4 in pneumococcal invasion.

Upstream stimulatory factor but not c-Myc enhances transcription of the human polymeric immunoglobulin receptor gene.

Secretory antibodies protect mucosal surfaces from ingested, inhaled and sexually transmitted pathogens. The polymeric immunoglobulin receptor (pIgR) transports antibodies across mucosal epithelia into external secretions. We and others have identified a region of the human polymeric immunoglobulin receptor gene (locus PIGR) that is sufficient for basal transcriptional activity. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper (bHLH/zip) family, was identified as a major regulatory element in the PIGR gene promoter. Transient transfection of PIGR promoter reporter plasmids in intestinal epithelial cell (IEC) lines suggested that the transcription factors upstream stimulatory factor (USF) and c-Myc may exert opposing effects on PIGR promoter activity. Mutations within and flanking the E-Box that favored USF binding enhanced promoter activity, while mutations that favored c-Myc binding reduced promoter activity. Ectopic expression of USF1 or USF2 enhanced PIGR promoter activity, while exogenous c-Myc did not. Electrophoretic mobility shift assays (EMSA) demonstrated that USF1 and USF2 bound to the E-Box motif as homo- and heterodimers. Chromatin immunoprecipitation (ChIP) demonstrated that USF proteins bind the PIGR promoter in vivo, which is enriched in acetylated histones. E-Box motifs are commonly observed in promoters of genes that are highly expressed in the human colon. Genes that are down-regulated in colorectal cancer, including PIGR, frequently have non-canonical E-Boxes, while genes that are up-regulated in colorectal cancer generally have canonical E-Boxes. The results of our experiments may shed light on the mechanisms of dysregulated expression of pIgR in inflammatory bowel disease and colorectal cancer, diseases associated with aberrant expression of c-Myc.

Characterization of the human polymeric immunoglobulin receptor (PIGR) 3'UTR and differential expression of PIGR mRNA during colon tumorigenesis.

The human cell lines VACO-235 and VACO-411 constitute a novel in vitro model of colon adenoma to carcinoma progression. By differential display RT-PCR we identified a transcript that is expressed in the parental nontumorigenic adenoma line (VACO-235E), but is not expressed in the tumorigenic daughter (VACO-235L) or granddaughter (VACO-411) lines. This cDNA represents a previously uncharacterized portion of the 3'UTR of human PIGR. Human PIGR mRNA was found to be highly expressed in normal colon epithelium, but was decreased in 6 of 8 colon tumors and was negligible in 8 of 10 colon tumor cell lines. We sequenced the entire 1.8 kb 3'UTR of human PIGR, and found it to contain multiple repetitive elements as well as elements that could affect the processing and stability of PIGR mRNA. We hypothesize that differential regulation of PIGR mRNA stability may contribute to its downregulation in colon cancer.