PTEN regulates colorectal epithelial apoptosis through Cdc42 signalling.

BACKGROUND: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear. METHODS: To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies. RESULTS: The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis. CONCLUSION: Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.

Expression of osteopontin coregulators in primary colorectal cancer and associated liver metastases.

BACKGROUND: A transcription regulatory complex (TRC) that includes Ets1, Ets2, PEA3 and ß-catenin/T-cell factors regulates osteopontin (OPN) that is implicated in colorectal cancer (CRC) dissemination. The consistency of OPN transcriptional control between primary CRC and metastases is unclear. This study investigates expression and prognostic significance of the OPN-TRC in primary human CRC and associated colorectal liver metastases (CRLM). METHODS: Osteopontin-TRC factors were assayed by digital microscopy in 38 primary CRCs and matched CRLM specimens and assessed against clinical prognosis. RESULTS: In primary CRC, OPN expression intensity correlated with that of its co-activators, PEA3 (r=0.600; P<0.01), Ets1 (r=0.552; P<0.01), Ets2 (r=0.521; P<0.01) and had prognostic significance. Osteopontin intensity in primary CRC inversely correlated with the interval between diagnosis and resection of CRLM. Overall OPN intensity was lower in CRLM than primary CRC and correlations with co-activators were weaker, for example, Ets1 (P=0.047), PEA3 (P=0.022) or nonsignificant (Ets2). The ratio of OPN expression in CRLM vs primary CRC had prognostic significance. CONCLUSION: This study supports transcriptional control of OPN by known coregulators in both primary and secondary CRC. Weaker associations in CRLM suggest involvement of other unknown factors possibly from the liver microenvironment or resulting from additional genetic or epigenetic changes that drive tumour metastatic capability in OPN transcriptional control.

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-γ can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-γ secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-γ responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-γ secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-γ secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-γ that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.

Rationale for cancer prevention strategies in high-risk ulcerative colitis.

Colorectal cancer (CRC) is the most feared long-term complication in patients with ulcerative colitis (UC) and Crohn's colitis. Surveillance by colonoscopy and serial biopsy is conducted to identify patients most likely to benefit from potentially curative surgery. Within this paradigm, patients with high grade dysplasia or early stage CRC typically undergo colectomy, while patients free of dysplasia continue within surveillance programmes. However, detection of dysplasia in colitis may be difficult. Underdiagnosis and undertreatment of dysplasia may be accompanied by 'interval cancers' after apparently negative colonoscopy, frustrating the goal of cancer prevention. In the absence of a best practice model, surgical decisions for effective cancer prevention and control can be aided by greater understanding of cancer biology, in particular the close relationship between processes of inflammation and neoplastic change. This review will summarise recent knowledge in this area and consider clinical variables of disease duration, severity and anti-inflammatory therapy against stepwise events of neoplastic transformation. Against this background, indications for surveillance and prophylactic colectomy in specific clinical situations will be discussed.

Active transport of benzo[a]pyrene in apical membrane vesicles from normal human intestinal epithelium.

Transport of the carcinogen benzo[a]pyrene in apical membrane vesicles (AMV) from normal human intestine, was investigated. Benzo[a]pyrene transport was found in AMV throughout the small intestine, but was greatest in colon. Evidence suggesting involvement of P-Glycoprotein (P-Gp), included (1) comparable transport of P-Gp substrate doxorubicin, (2) transport stimulation by ATP and (3) transport suppression by the P-Gp inhibitor, verapamil.

Dipyridamole potentiates antipurine antifolate activity in the presence of hypoxanthine in tumor cells but not in normal tissues in vitro.

The cytotoxicity of the antifolate inhibitors of de novo purine biosynthesis, lometrexol (LTX) and LY309887, can be abolished by hypoxanthine (HPX) salvage. The nucleoside transport inhibitor, dipyridamole (DP) can prevent HPX rescue from LTX growth inhibition in a cell line-specific manner. The studies described here have shown that, excluding colon and hematological malignancies, DP prevents HPX rescue from LTX growth inhibition in approximately one-third of cell lines with otherwise limited tissue specificity. The clinical dose-limiting toxicities of antipurine antifolates are to the bone marrow and gastrointestinal tract. In vitro models of these normal tissues were established, and the effect of DP on HPX rescue from LY309887 treatment was studied. Growth inhibition assays are not feasible in these primary cultures; therefore, an alternative assay, cellular ATP depletion, was validated in four tumor cell lines as a marker of de novo and salvage purine synthesis. In LY309887-treated cells, DP prevented HPX-mediated maintenance of ATP levels only in cell lines in which DP inhibited HPX rescue from antifolate cytotoxicity. Hence, ATP depletion is a reliable indicator of sensitivity of HPX transport to DP when direct cell growth measurement is impractical. In primary cultures of human hematopoetic progenitor cells and mouse small intestine, coincubation with HPX prevented LY309887-mediated ATP depletion, which was not blocked by DP. These data suggest that DP would not prevent HPX rescue from antipurine antifolate growth inhibition in sensitive normal tissues, whereas activity against certain solid human tumors would be maintained.

Phenol sulphotransferase SULT1A1*1 genotype is associated with reduced risk of colorectal cancer.

Sulphation is an important detoxification pathway for numerous xenobiotics; however, it also plays an important role in the metabolism and bioactivation of many dietary and environmental mutagens, including heterocyclic amines implicated in the pathogenesis of colorectal and other cancers. A major sulphotransferase (SULT) enzyme in humans, SULT1A1, is polymorphic with the most common variant allele, SULT1A1*2, occurring at a frequency of about 32% in the Caucasian population. This allele codes for an allozyme with low enzyme activity and stability compared to the wild-type (SULT1A1*1) enzyme, and therefore SULT1A1 genotype may influence susceptibility to mutagenicity following exposure to heterocyclic amines and other environmental toxins. Previously, a significant association of SULT1A1*1 genotype with old age has been observed, suggesting a 'chemoprotective' role for the high-activity phenotype. Here we have compared the frequencies of the most common SULT1A1 alleles in 226 colorectal cancer patients and 293 previously described control patients. We also assessed whether SULT1A1 genotype was related to various clinical parameters in the patient group, including Duke's classification, differentiation, site, nodal involvement and survival. There was no significant difference in allele frequency between the control and cancer patient populations, nor was there a significant association with any of the clinical parameters studied. However, when the age-related difference in allele frequency was considered, a significantly reduced risk of colorectal cancer (odds ratio = 0.47; 95% confidence interval = 0.27-0.83; P = 0.009), was associated with homozygosity for SULT1A1*1 in subjects under the age of 80 years. These results suggest that the high activity SULT1A1*1 allozyme protects against dietary and/or environmental chemicals involved in the pathogenesis of colorectal cancer.

Zamifenacin (UK-76, 654) a potent gut M3 selective muscarinic antagonist, reduces colonic motor activity in patients with irritable bowel syndrome.

BACKGROUND: Zamifenacin is a new potent gut M3 selective muscarinic antagonist developed for possible use in irritable bowel syndrome. METHODS: In this multicentre, double-blind, parallel group, placebo-controlled study, the effect of a single dose of zamifenacin 10 mg or 40 mg on both fasting (30 min) and fed (60 min) colonic motor activity was assessed in 36 patients with irritable bowel syndrome (aged 25-68 years; 19 male). Colonic motility was recorded using a five-channel solid-state catheter introduced by colonoscopy to a depth of 35 cm in an unprepared colon. RESULTS: Zamifenacin 40 mg profoundly reduced colonic motility, particularly after the meal (P < 0.05). This was reflected by a significant reduction in the mean amplitude of contractions, number of contractions, percentage duration of contractions, activity index and the motility index (P < 0.05). A smaller reduction in all the motility parameters was obtained with 10 mg zamifenacin, but these changes were not statistically significant. Three patients each on placebo and zamifenacin reported side-effects, but these were mild and transient. CONCLUSION: A single 40 mg dose of zamifenacin significantly reduces colonic motility in irritable bowel syndrome patients without significant antimuscarinic effects. The results of this study confirm that the concept of developing selective antimuscarinic agents may be a promising approach to the treatment of irritable bowel syndrome. Not only would such compounds benefit from not having the usual side-effects of anticholinergics but they might also offer much more in the way of dose flexibility.

Progressive morphogenesis in vivo after transplantation of cultured small bowel epithelium.

An experimental model for the primary culture and transplantation of late foetal rat small intestinal epithelium is described. Multicellular aggregates of mucosal epithelium containing pre-crypt proliferative cells were isolated from 20-day foetal rat intestine by enzymatic disaggregation. Cellular aggregates, which we refer to as "epithelial organoids," attached readily in culture, proliferated, and spread to produce coalescing colonies within 10 days. Enterocytes were maintained in culture for 3 days, removed as cell sheets, and incubated overnight with foetal mesenchyme. Fourteen recombinant preparations were then grafted to the renal subcapsular space of adult nude mice. Four of six grafts retrieved after 1 wk had developed. Histology demonstrated the formation of simple tubular structures lined by a polarized columnar epithelium. At 14 days, two of eight grafts had developed and demonstrated temporal progression of morphogenesis. Histology showed rudimentary crypts and villi lined by different epithelial cell types, including enterocytes and goblet cells. Small bowel proliferative cells within "epithelial organoids" from 20-day foetal intestine, may be maintained in primary culture for up to four days. After short term primary culture, these proliferative cells retain the capacity for progressive organotypic morphogenesis and pluripotent cytodifferentiation, after transplantation to adult recipients.

Does neomucosa induced by small bowel stem cell transplantation have adequate function?

Small intestinal epithelium digests and absorbs nutrients. Crypt stem cell transplantation can generate neomucosa with normal morphology, but the digestive and absorptive capacities of this neomucosa are unknown. This study evaluates stem cell induced neomucosal brush border digestive enzyme activity and nutrient transport function. Rodent small intestinal epithelial stem cells were isolated by enzymatic digestion, then grafted to inbred recipients. Grafts were retrieved at 25 days, and apical brush border membrane vesicles prepared for quantitative assays. Neomucosal lactase, sucrase, aminopeptidase N, and alkaline phosphatase activity was determined by incubation with enzyme specific substrate. Neomucosal sodium dependent D-glucose transport was evaluated by incubation with D-[U-14C] glucose. Comparative assays were performed in age-matched control intestine. Neomucosal digestive enzyme activities and D-glucose transport were all similar in neomucosa and control small intestine.

Colonic mucosal replacement by syngeneic small intestinal stem cell transplantation.

A novel method of colonic mucosal replacement by transplantation of disaggregated small intestinal epithelium is described. Thirty-one inbred rats had the ascending colon isolated, and surgical mucosectomy was performed on the "free" loop. Epithelial cell aggregates were isolated from postnatal small intestine using collagenase and dispase digestion, then 20 microL of the cell suspension was "seeded" over the denuded colonic muscle of 25 recipient rats. Six control rats had surgical mucosectomy only. All loops were retrieved after 14 days for histologic examination. Stem cell lineage studies were used with selective staining protocols to identify enterocytes, goblet cells, entero-endocrine cells, and Paneth cells. A neomucosa with typical small bowel morphology including crypts and villi and all four stem cell lineages was regenerated by transplanted cells on the colonic muscle in 19 of 25 (76%) recipients. Control loops showed no epithelial regrowth confirming total mucosectomy. With appropriate stromal support, transplanted small intestinal stem cells have the capacity to re-epithelialize denuded colonic muscle with small bowel neomucosa.

Vascular endothelial growth factor levels in serum and plasma following esophageal cancer resection--relationship to platelet count.

In patients with cancer circulating vascular endothelial growth factor (VEGF) may be tumor-derived and have prognostic significance. Activated platelets may also be a source of VEGF, releasing it in serum formation. Debate exists as to whether serum or plasma VEGF (S-VEGF, P-VEGF) is the most appropriate surrogate marker of tumor angiogenesis. As healing wounds produce VEGF that can spill over into the circulation, we aimed to investigate the potential confounding effects of cancer surgery on both perioperative S-VEGF and P-VEGF levels and to evaluate their relationship with platelet count. S-VEGF, P-VEGF and platelet counts were measured in 23 patients undergoing esophageal cancer resection. Samples were taken preoperatively and six weeks following surgery. Seven patients were also sampled on postoperative days 1, 5 and 10. VEGF was assayed using a commercial enzyme linked immunosorbent assay. S-VEGF and P-VEGF both rose after surgery (S-VEGF; day 5: 1017 [446-1224] pg/mL and day 10: 1231 [626-2046] pg/mL versus pre-op: 329 [189-599] pg/mL. P-VEGF; day 1: 55 [46-104] pg/mL and day 10: 58 [20-154] pg/mL versus pre-op: 23 [13-46] pg/mL), falling towards preoperative levels by six weeks. Platelet count correlated with S-VEGF (rho=0.281; p<0.05, Spearman's rank) and P-VEGF (rho=0.330; p<0.01, Spearman's rank). Platelets may contribute to VEGF levels in plasma as well as in serum. The effects of surgery on S-VEGF or P-VEGF levels are mainly transient. Care must be exercised when interpreting circulating VEGF levels in the early postoperative period.

Age-dependent change of metabolic capacity and genotoxic injury in rat intestine.

The ontogeny of intestinal phase I and II xenobiotic metabolising enzymes and influence on susceptibility to genotoxic injury, are unclear. This study assessed expression of cytochrome P450 monooxygenases (CYP1A, CYP2B, CYP2C, CYP3A, CYP4A), glutathione-S-transferase (GSTA1/2, GSTA3, GSTA4, AND GSTM1), and uridine diphosphate glucuronosyl transferase (UGT) in rat intestine, between fetal life and maturity. Enzyme induction and DNA adduct formation were assessed after 3-methylcholanthrene (MC) exposure. Untreated rat intestine expressed CYP2B, GSTA1/2, GSTA4 and UGT at all stages of maturation, although CYP2B and GSTA1/2 increased in postnatal life. MC induced new expression of CYP1A, GSTA3 and enhanced expression of GSTA1/2 and UGT. Age-dependent differences of enzyme induction and DNA adduct formation between pre- and postnatal intestine and during postnatal maturation, were observed. Rat intestinal epithelium shows variable competence for MC metabolism and sustains disparate levels of DNA adducts during pre- and postnatal development.

Gallbladder function and fasting enterogastric bile reflux.

The relationship between gallbladder function and duodenogastric reflux of bile has been investigated in the fasting phase by 99Tcm-EHIDA biliary scintigraphy in 370 patients with abdominal pain. This isotope is cleared from the blood-pool by hepatocellular function and is excreted into the biliary tract. Obstruction of the common bile duct was found in 79 patients, leaving 291 who were evaluable for duodenogastric bile reflux. Gallblader function was present in 169 and absent in 122 patients. Duodenogastric reflux of bile was observed in 82 patients, 10 of whom had had previous gastric surgery. Only 298 of 169 (17%) patients with a functioning gallbladder showed evidence of enterogastric bile reflux compared to 43 of 122 (35%) with absent gallbladder function (p less than 0.005 by Chi Square). These results suggest that continuous bile flow into the duodenum in the absence of a functioning gallbladder facilitates duodenogastric reflux.

Circulating neutrophil priming and systemic inflammation in limb ischaemia-reperfusion injury.

BACKGROUND: Recruitment and activation of neutrophils is a key step in the development of local and systemic injury in lower limb ischaemia-reperfusion. We hypothesis that increased circulating neutrophil priming is responsible for systemic inflammation. METHODS: Anaesthetised ventilated swine (n = 6 per group) underwent mid-line laparotomy and were randomised to control group or bilateral external iliac artery occlusion for two hours followed by two and a half hours reperfusion (I/R group). Using luminol, respiratory burst activity was assayed with a BioOrbit Luminometer to detect whole blood chemiluminescence (CL) by stimulation with phorbol 1,2-myristate 1,3-acetate (PMA) in the absence or presence of tumour necrosis factor (TNF) respectively. PMN priming is expressed as the ratio of whole blood CL in the presence of TNF to that without. We measured plasma interleukin(IL)-6 and tumour necrosis factor alpha by bioassay as a measure of systemic inflammation. The alveolar-arterial (A-a) gradient was measured using the formula [(A-a)gradient = fraction inspired O2 x 710-(arterial pCO2/0.8)-arterial pO2], it is a measure of lung function, a large gradient being indicative of impaired oxygen transport and hence lung injury. RESULTS: Lower limb I/R caused significantly greater PMN priming, 0.83 +/- 0.14, compared to control group, 0.22 +/- 0.04, (p < 0.001). Plasma IL-6, a reliable indicator of systemic inflammation, was significantly increased in I/R group after two and a half hours of reperfusion, 1295.0 (833.9-2073.0) pg/L, compared to control, 382.9 (367.4-568.3) pg/L, (p < 0.005). Plasma tumour necrosis factor alpha was significantly elevated after one hour of reperfusion in the I/R group, 86.8 (48.7-106.6) pg/ml, compared to the control group, 32.7 (0.9-42.8) pg/ml, (p < 0.01). (A-a) gradient was significantly increased after IRI, 407.97 +/- 53.13, compared to the control, 183.19 +/- 45.75, (p < 0.005). Mean pulmonary artery pressure was significantly greater after IRI, 38.80 +/- 4.87 mmHg, compared to control, 27.86 +/- 1.92 mmHg, (p < 0.005). Data represents mean +/- standard error mean or median (interquartile range), statistical comparisons using one-way Anova with Student's "t"-test and Kruskall-Wallis Anova with the Mann-Whitney U test. CONCLUSIONS: Priming of neutrophils increases their circulating respiratory burst activity and ability to induce tissue injury. Systemic PMN priming during hind limb ischaemia-reperfusion injury is associated with the systemic inflammatory response syndrome.

Computerised method for acquisition and display of gastrointestinal motility data.

A computerised system is developed for the acquisition and display of gastrointestinal motility data which utilises a purpose developed software program called 'PC-motil', running on an IBM compatible microcomputer. 'PC-motil' displays data during collection, writes data to disk file and compresses all data at the end of a study on to a single monitor screen for convenient overview. Any area of interest, in single or multiple channels, may be selected and expanded for detailed examination. This system is tested by the recording of gastric and jejunal motility patterns of 11 healthy volunteers in fasting and fed states. All antral and jejunal migrating motor complexes (MMCs) in fasting studies, as well as all fed motility patterns were recognisable in both 'compressed' and 'expanded' form. The reproduction of motility patterns by the computer based system was indistinguishable from that of a conventional analogue chart recorder. This computerised system provides a convenient and cost-effective means of acquisition, storage and display of motility data in digital form.

Prospective study of a planned approach to the surgical management of bleeding oesophageal varices.

A policy of joint management of patients with acute variceal bleeding was constructed and applied prospectively to 37 consecutive patients. According to the protocol, every patient underwent an initial joint assessment by a physician and a surgeon soon after admission and resuscitation, which included age, previous history, physical examination and biochemical profile. Pugh's modification of Child's scoring system was used to assess the severity of the underlying hepatic disorder. Patients in groups A and B who were less than 70 years of age were treated by shunt surgery carried out during the same admission. Patients outwith this category were managed conservatively and a blood transfusion limit (10 units) imposed unless the clinical picture subsequently improved or the liver function was good but the patient was not eligible for surgery because of age. Twenty seven patients underwent shunt surgery, the most frequent procedure being a Warren shunt (n = 21). The average hospital stay in the surgically treated group was 18 days. The 30 day operative mortality was 15%. Two further deaths in the shunted group occurred during the follow-up period (mean = 30 months, range = 6-97 months). Seventeen patients in the shunted group remain well but three have required further therapy because of rebleeding episodes (two with sclerotherapy, one by surgery). Two patients who went abroad after recovery from their shunt surgery have been lost to follow up. The survival in the conservatively treated group was short and recurrent bleeding common. This experience indicates that an initial joint assessment using established criteria reliably identifies the poor prognosis group of patients with variceal haemorrhage and allows the rationalisation of the hospital resources.Early and expeditious selective shunt surgery in the good risk category carries an acceptable mortality and is cost effective in the long term.

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy.

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ), cultures were treated with the PPARγ ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPARγ antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPARγ ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression.

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system.