Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

Oncogenic K-RAS subverts the antiapoptotic role of N-RAS and alters modulation of the N-RAS:gelsolin complex.

Activating mutations in members of the RAS family of genes are among the most common genetic events in human tumorigenesis. Once thought to be functionally interchangeable, it is increasingly recognized that the classical members of this protein family (H-RAS, N-RAS and K-RAS4B) exhibit unique and shared functions that are highly context-dependent. Herein, we demonstrate that the presence of an oncogenic KRAS allele results in elevated levels of GTP-bound N-RAS (N-RAS.GTP) in two human colorectal cancer cell lines, HCT 116 and DLD-1, compared to their isogenic counterparts in which the mutant KRAS allele has been disrupted by homologous recombination. N-RAS subserves an antiapoptotic role in cells expressing wild-type K-RAS; this function is compromised, however, by the presence of mutant K-RAS, and these cells display increased sensitivity to apoptotic stimuli. We additionally identify a physical interaction between N-RAS and gelsolin, a factor that has been shown to promote survival and show that the N-RAS:gelsolin complex is modulated differently in wild-type and mutant K-RAS environments following apoptotic challenge. These findings represent the first biochemical evidence of a functional relationship between endogenous RAS proteins and identify a dynamic physical interaction between endogenous N-RAS and gelsolin that correlates with survival.

Over-expression of cyclin D1 regulates Cdk4 protein synthesis.

Increased Cdk4 expression occurs coincident with over-expression of cyclin D1 in many human tumours and tumourigenic mouse models. Here, we investigate both in vivo and in vitro the mechanism by which Cdk4 expression is regulated in the context of cyclin D1 over-expression. Cdk4 mRNA levels in cyclin D1-over-expressing tissue and cultured cells were unchanged compared with controls. In contrast, Cdk4 protein levels were increased in cyclin D1-over-expressing tissue and cells versus their respective controls. This increase was not due to altered protein stability, but appeared to be due to an increase in Cdk4 protein synthesis. We also performed immunoprecipitation and in vitro kinase assays to demonstrate an increase in cyclin D1-Cdk4 complex formation and associated kinase activity. Blocking cyclin D1 expression resulted in diminished Cdk4 protein but not mRNA levels. These findings suggest a mechanism by which Cdk4 expression is increased in the context of cyclin D1 over-expression during tumourigenesis.

Expression of interleukin-6, leukemia inhibitory factor and their receptors by colonic epithelium and pericryptal fibroblasts.

BACKGROUND AND AIM: The cellular configuration of the human colon suggests a predetermined organization that creates specific microenvironments. The role of pericryptal fibroblasts in this microenvironment has been the subject of considerable speculation. This study examined the expression of growth factors and their receptors by colonic crypt epithelium and pericryptal fibroblasts. METHODS AND RESULTS: Pericryptal fibroblast cells were isolated and cultured from decrypted human colonic mucosa. The pericryptal fibroblast cells expressed messenger RNA (mRNA) for interleukin-6 (IL-6), leukemia inhibitory factor (LIF), LIF receptor alpha, and the common coreceptor glycoprotein 130 (GP130), but not the IL-6 receptor alpha. Interleukin-6 protein expression was confirmed by the analysis of conditioned medium and immunohistochemistry. In comparison, normal colonic epithelial cells express mRNA for LIF but not IL-6 as well as the receptors for GP-130, IL-6 receptor alpha but not LIF receptor alpha. As cultures of normal human colonic epithelial cells were not available, the conditioned medium was assayed from established colon carcinoma cell lines and demonstrated a secretion of LIF but not IL-6 protein. CONCLUSION: The expression of reciprocal cytokine and receptor expression suggest that there is a paracrine relationship between pericryptal fibroblasts and colonic epithelium.

The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors.

Phosphatidylinositol 3'-kinases (PI3ks) are a family of lipid kinases that play a crucial role in a wide range of important cellular processes associated with malignant behavior including cell growth, migration, and survival. We have used single-strand conformational polymorphism/heteroduplex analysis to demonstrate the presence of somatic mutations in the gene for the p85alpha regulatory subunit of PI3k (PIK3R1) in primary human colon and ovarian tumors and cancer cell lines. All of the mutations lead to deletions in the inter-SH2 region of the molecule proximal to the serine608 autoregulatory site. Expression of a mutant protein with a 23 amino acid deletion leads to constitutive activation of PI3k providing the first direct evidence that p85alpha is a new oncogene involved in human tumorigenesis.

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line.

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.

Insulin-like growth factor (IGF)-binding protein-6 inhibits IGF-II-induced but not basal proliferation and adhesion of LIM 1215 colon cancer cells.

IGF-II is an autocrine growth factor for many colon cancer cells. This study aimed to determine the role of IGF-II in proliferation and adhesion of LIM 1215 colon cancer cells. RT-PCR demonstrated expression of IGF-I and IGF-II mRNA. Addition of IGF-I or -II increased monolayer proliferation in a dose-dependent manner. Although addition of IGFBP-6 had no effect on basal proliferation, coincubation of IGFBP-6 decreased IGF-II but not IGF-I-induced proliferation. Colony formation in agar was increased by IGF-II, an effect inhibited by coincubation with IGFBP-6. IGFBP-6 alone significantly decreased colony formation. Preincubation of cells with IGF-II increased adhesion to type IV collagen, fibronectin and laminin. IGFBP-6 had no effect on basal cell adhesion but completely inhibited the effects of IGF-II. LIM 1215 colon cancer cells are therefore IGF-responsive but IGF-II is not a major autocrine factor for these cells in monolayer, suggesting heterogeneity between colon carcinoma cell lines with respect to the role of the IGF system.

PMC42, a novel model for the differentiated human breast.

Cultured human breast carcinoma cell lines are important models for investigating the pathogenesis of breast cancer. Their use, however, is limited because of loss of expression of breast-specific markers and the development of a dedifferentiated phenotype after continuous culture. PMC42 is a unique human breast carcinoma line, previously shown to express secretory and myoepithelial markers. We have induced PMC42 cells to form hollow organoids in culture, similar to in vivo breast structures, using a combination of hormones including estrogen, progesterone, dexamethasone, insulin, and prolactin in combination with a permeable extracellular matrix. The organoids comprised polarized cells located around a central lumen. Expression of beta-casein was demonstrated in cells within organoids using reverse transcriptase-polymerase chain reaction, Western blot analysis, and confocal immunofluorescence. In this in vitro system, milk-specific gene expression was induced through hormone and matrix interactions which may be similar to those operating in vivo. PMC42 is a novel model for investigations into the molecular mechanisms of carcinogenesis and differentiation in the human breast.

Conditionally immortalized mouse hepatocytes for use in liver gene therapy.

BACKGROUND AND AIMS: The use of hepatocytes for gene therapy is limited by the difficulty of maintaining and altering primary liver cells in culture. A conditionally immortalized mouse hepatocyte cell line has been developed which can be passaged indefinitely at the permissive temperature (33 degrees C), but fails to proliferate and dies at the non-permissive temperature (39 degrees C) in vitro. METHODS: Hepatocytes were harvested from a 6 week-old male transgenic mouse ('immortomouse') carrying a thermolabile SV40 Large T gene, using a modified two-step collagenase perfusion method, and serially passaged at 33 degrees C for more than 1 year. To assess the ability of immortohepatocytes to engraft and populate mouse liver, cells were infused into partially hepatectomized congenic mice via the portal vein (n = 10) or the spleen with (n = 2) and without (n = 2) partial hepatectomy. The ability to transfect immortohepatocytes was assessed using the reporter gene enhanced green fluorescent protein (EGFP). RESULTS: All immortohepatocytes in culture stained positive by immunohistochemistry for the hepatocyte markers albumin, AFP, CK8 and CK18. In early cultures a proportion of cells also stained strongly for the biliary epithelial markers CK7 and CK19. Late cell cultures were negative for M2PK and CK7 and stained variably with anti-CK19 antibodies. Cells transferred to the non-permissive temperature of 39 degrees C ceased proliferation and died within 1 week in vitro. Large T DNA was detected in the liver of all postoperative mice up to 2 weeks post-hepatocellular transplantation via PCR and Southern blot analysis. The immortohepatocytes were easily transfected with a reporter gene. CONCLUSIONS: Immortohepatocytes can survive in vivo after transfer to liver, and will be useful as a model for hepatic gene therapy.

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells.

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.

Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans.

BACKGROUND & AIMS: The factors controlling the proliferation and differentiation of the colonic mucosa are unknown and have proved difficult to identify mainly because of a lack of in vitro methods for studying the proliferative cells of the mucosa. METHODS: We have developed a novel method of preparing a viable single-cell suspension from isolated crypts and cloning these single cells. RESULTS: We have obtained clonogenic growth from this single-cell suspension with an average of 1 colony per 10(5) cells in control cultures. Addition of conditioned medium from the LIM1863 colon carcinoma cell line increased the mean colony number to 11 +/- 3 per 10(5) cells. The cells forming the colonies are still viable after 4 weeks in culture. The epithelial nature of the cells was confirmed by ultrastructural and immunohistochemical methods with staining for keratin 8 and 18 and anti-human epithelial membrane-specific antigen and a positive result on polymerase chain reaction for keratin 19. CONCLUSIONS: We have successfully cloned single cells from disaggregated colonic crypts from both human and murine colonic mucosa. We have also demonstrated the presence of an active clonogenic factor in the conditioned medium of a colon carcinoma cell line. Assays show that the clonogenic activity in the conditioned medium is not caused by the presence of any of the epidermal growth factor family of growth factors. This is the first report of a clonogenic assay for epithelial cells of normal colonic mucosa.

c-Myb down-regulation is associated with human colon cell differentiation, apoptosis, and decreased Bcl-2 expression.

c-myb is expressed in human and murine colonic mucosa and elevated expression occurs in premalignant adenomatous polyps and carcinomas. c-Myb is required for colon cell proliferation, and there is evidence of c-myb down-regulation during differentiation. Recently, c-myb has been implicated in hematopoietic cell survival via regulation of bcl-2 gene expression. However, c-myb expression during terminal differentiation and apoptosis in the colonic crypt has not been examined. The experiments in this study examine the spatial and temporal expression of c-Myb protein in vivo using human colonic crypt sections and in vitro in human colon tumor cell lines undergoing butyrate-induced differentiation and apoptosis. Electron microscopy, together with molecular and biochemical analysis, was used to define the differentiation status of the cells. Results demonstrate a decrease in c-Myb expression during the commitment of cells to differentiation and apoptosis. Decreased levels of c-Myb are accompanied by a decrease in Bcl-2. These data suggest that the transcription factor c-Myb has a role in regulating the balance between proliferation, differentiation, and apoptosis in the colonic crypt. Furthermore, elevated c-Myb levels in colon tumor cells may lead to persistent bcl-2 expression, thus protecting tumor cells from programmed cell death.

Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells.

Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.

Rapid diagnostic test for hereditary nonpolyposis colon cancer kindred using polymerase chain reaction.

PURPOSE: We have identified a mutation in the hMLH1 gene from the proband of a hereditary nonpolyposis colorectal cancer kindred. We wished to develop a rapid test for this specific mutation to facilitate screening of other family members. METHOD: An allele-specific polymerase chain reaction strategy was used to detect a T insertion at the + 3 splice site post exon 9 in the hMLH1 gene. The test was evaluated on DNA in which the mutation status was known. RESULTS: A 130-base pair fragment was reliably amplified using the allele-specific polymerase chain reaction. The test is able to identify the mutant allele and to distinguish between normal, carriers (heterozygous), and tumor DNA samples. The mutant allele is not present in an unrelated hereditary nonpolyposis colorectal cancer cell line or in a sample of the normal population (n=49). CONCLUSIONS: This is a simple, rapid test that can determine carrier status in the members of a kindred at risk for this mutation. This mutation is unlikely to be a polymorphism. This test may now be evaluated in a clinical setting.

The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily.

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.

Colonic expression of c-myb is initiated in utero and continues throughout adult life.

c-myb expression is an indicator of hemopoietic cell proliferation and its down-regulation occurs as an early event in cellular differentiation in vitro. We have previously demonstrated c-Myb protein expression in normal adult colon and tumor-derived colonic cell lines and have also shown that the proliferation of colon carcinoma cell lines is c-myb dependent. This study used the techniques of RNase protection and immunohistochemistry to define c-myb mRNA and protein expression in the normal adult mouse and mouse embryos, with special focus on the colon. These experiments revealed wide-spread c-myb mRNA expression in mouse embryos including nonhemopoietic tissues, and developmental time course studies revealed alterations in organ-specific c-myb mRNA expression. Immunohistochemical studies confirmed the embryonal expression of c-Myb in concordance with the mRNA data as well as c-Myb expression throughout the length of the adult mouse colonic crypt. In contrast, staining for the proliferating cell nuclear antigen was confined to the lower one third of the crypt. In addition, c-Myb staining extended beyond that of the proliferating cell nuclear antigen within the germinal centers of the spleen. These data suggest that c-myb: (a) has a role in the embryonic development of nonhemopoietic organs; (b) plays a specific role in the biology of the normal adult colonic crypt; and (c) expression is not inextricably linked to proliferation in vivo.

Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.

Effects of vesicocolic anastomosis on the proliferation of colonic mucosa in a rat model.

The effects of diverting the urinary stream into a defunctioned segment of the distal colon has been studied in a rat model. Distal rat colon was defunctioned using the Hartmann's procedure and a vesicocolic anastomosis was performed 14 days later. The distal colon was harvested after 10 days following the administration of a vinblastine mitotic block either 1 or 3 h before death. Both the mean crypt cell proliferation and the crypt length were increased significantly in the colonic mucosa exposed to urine when compared with the control defunctioned colon or to the functional unexposed proximal colon. An active fraction was obtained from human urine by elution from a diethylaminoethyl sepharose column using 1 mol/L NaCl. This fraction was administered intraluminally to defunctioned rat colons using Alzet minosmotic pumps. In these animals the crypt cell production rate was significantly increased compared with the control animals. Although the crypt length did not increase significantly in these animals the atrophy normally seen in defunctioned colonic mucosa did not occur. The identity of the active molecule in this urine fraction is still being determined.

Effect of TGF-beta on differentiated organoids of the colon carcinoma cell line LIM 1863.

The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.