Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

ATPase activity and ATP-dependent conformational change in the co-chaperone HSP70/HSP90-organizing protein (HOP).

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.

An open-label prospective randomized multicenter study of intensive versus weekly granulocyte and monocyte apheresis in active crohn's disease.

BACKGROUND: Granulocyte and monocyte adsorptive apheresis (GMA) has shown efficacy in patients with active Crohn's disease (CD). However, with routine weekly therapy, it may take several weeks to achieve remission. This study was performed to assess clinical efficacy and safety of intensive GMA in patients with active CD. METHODS: In an open-label, prospective, randomized multicentre setting, 104 patients with CD activity index (CDAI) of 200 to 450 received intensive GMA, at two sessions per week (n = 55) or one session per week (n = 49). Clinical remission was defined as a CDAI score <150. Patients in each arm could receive up to 10 GMA sessions. However, GMA treatment could be discontinued when CDAI decreased to <150 (clinical remission level). RESULTS: Of the 104 patients, 99 were available for efficacy evaluation as per protocol, 45 in the weekly GMA group, and 54 in the intensive GMA group. Remission was achieved in 16 of 45 patients (35.6 %) in the weekly GMA and in 19 of 54 (35.2 %) in the intensive GMA (NS). Further, the mean time to remission was 35.4 ± 5.3 days in the weekly GMA and 21.7 ± 2.7 days in the intensive GMA (P = 0.0373). Elevated leucocytes and erythrocyte sedimentation rate were significantly improved by intensive GMA, from 8005/µL to 6950/µL (P = 0.0461) and from 54.5 mm/hr to 30.0 mm/hr (P = 0.0059), respectively. In both arms, GMA was well tolerated and was without safety concern. CONCLUSIONS: In this study, with respect to remission rate, intensive GMA was not superior to weekly GMA, but the time to remission was significantly shorter in the former without increasing the incidence of side effects. UMIN registration # 000003666.

The components of the AhR-molecular chaperone complex differ depending on whether the ligands are toxic or non-toxic.

The aryl hydrocarbon receptor (AhR) forms a complex with the HSP90-XAP2-p23 molecular chaperone when the cells are exposed to toxic compounds. Recently, 1,4-dihydroxy-2-naphthoic acid (DHNA) was reported to be an AhR ligand. Here, we investigated the components of the molecular chaperone complex when DHNA binds to AhR. Proteins eluted from the 3-Methylcolanthrene-affinity column were AhR-HSP90-XAP2-p23 complex. The AhR-molecular chaperone complex did not contain p23 in the eluents from the DHNA-affinity column. In 3-MC-treated cells, AhR formed a complex with HSP90-XAP2-p23 and nuclear translocation occurred within 30 min, while in DHNA-treated cells, AhR formed a complex with AhR-HSP90-XAP2, and translocation was slow from 60 min. Thus, the AhR activation mechanism may differ when DHNA is the ligand compared to toxic ligands.

Traditional Herbal Medicine, Rikkunshito, Induces HSP60 and Enhances Cytoprotection of Small Intestinal Mucosal Cells as a Nontoxic Chaperone Inducer.

Increasing incidence of small intestinal ulcers associated with nonsteroidal anti-inflammatory drugs (NSAIDs) has become a topic with recent advances of endoscopic technology. However, the pathogenesis and therapy are not fully understood. The aim of this study is to examine the effect of Rikkunshito (TJ-43), a traditional herbal medicine, on expression of HSP60 and cytoprotective ability in small intestinal cell line (IEC-6). Effect of TJ-43 on HSP60 expression in IEC-6 cells was evaluated by immunoblot analysis. The effect of TJ-43 on cytoprotective abilities of IEC-6 cells against hydrogen peroxide or indomethacin was studied by MTT assay, LDH-release assay, caspase-8 activity, and TUNEL. HSP60 was significantly induced by TJ-43. Cell necrosis and apoptosis were significantly suppressed in IEC-6 cells pretreated by TJ-43 with overexpression of HSP60. Our results suggested that HSP60 induced by TJ-43 might play an important role in protecting small intestinal epithelial cells from apoptosis and necrosis in vitro.

Colonic mucosal lesions associated with low-dose aspirin: a case control study.

OBJECTIVE: Low-dose aspirin (LDA) is widely used because it reduces the risk of vascular events in patients with atherosclerosis. Recently, there has been a substantial increase in prescriptions for LDA. We analyzed the risk of colonic mucosal lesions associated with the long-term use of LDA. MATERIAL AND METHODS: Among Japanese patients who underwent a colonoscopy between January 2004 and December 2006, 199 colitis cases and 5764 non-colitis controls were identified after excluding 749 patients based on study criteria. The history of LDA use was compared between the cases and controls and the multivariate (age-, sex- and underlying diseases-) adjusted odds ratio (OR) was estimated using a multiple logistic regression model. RESULTS: The adjusted OR for colonic mucosal lesions associated with LDA use versus non-use was 1.45 [95% confidence interval (CI), 0.87-2.42; p = 0.152]. In terms of gender differences, the OR for LDA-induced colitis in females was significantly increased at 2.55 (95% CI, 1.31-4.94; p = 0.006) but was not significantly increased in males at 0.70 (95% CI, 0.34-1.45; p = 0.334). CONCLUSIONS: In females, LDA increased the risk of colonic mucosal lesions, suggesting that LDA may contribute to the pathogenesis of colonic ulceration or colitis. Therefore, it is essential that prescribing physicians be aware of the risk of LDA-induced colonic lesions.

Evidence for enhanced cytoprotective function of HSP90-overexpressing small intestinal epithelial cells.

BACKGROUND: In the clinical field, increasing incidence of small intestinal ulcers associated with nonsteroidal anti-inflammatory drugs (NSAIDs) has become a topic with the advances of capsule endoscopy and balloon enteroscopy technology for the detection of small intestinal lesions. However, the pathogenesis of NSAID-induced mucosal damage, defensive mechanism of intestinal epithelial cells, and therapy for small intestinal mucosal lesion have not been fully understood. Heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. Since the function of HSP90 in the intestinal epithelial cells has not been well investigated, we examined the cytoprotective ability of HSP90-overexpressing small intestinal epithelial cells against hydrogen peroxide-induced or indomethacin-induced cell damage. METHODS: cDNA of human HSP90 gene was transfected to rat small intestinal epithelial cells (IEC-6 cells), and HSP90-overexpressing cells (IEC-6-90 cells) were selected and cloned. Anti-necrotic abilities and anti-apoptotic abilities of IEC-6-90 cells were compared with IEC-6-mock cells (transfected with vector alone). To examine the specific contribution of HSP90 on cytoprotection of IEC-6-90 cells, cytoprotective ability of IEC-6-90 cells was analyzed with or without pretreatment with functional inhibitor of HSP90, geldanamycine analog, followed by hydrogen peroxide-challenge or indomethacin-challenge. RESULTS: Hydrogen peroxide-induced or indomethacin-induced cell necrosis and apoptosis were significantly suppressed in IEC-6-90 cells. The cytoprotective ability of IEC-6-90 cells was suppressed by HSP90 inhibitor. CONCLUSIONS: Our results suggest that HSP90 might play an important role in protecting small intestinal epithelial cells from hydrogen peroxide-induced or indomethacin-induced cell injury in vitro, and raised the possibility of protection of small intestinal epithelial cells by manipulation of HSP90 expression.

Establishment of a reflux esophago-laryngitis model in rats.

BACKGROUND: To investigate the pathophysiology of reflux laryngitis, an experimental model is required. AIM: The aim of this study is to establish an animal model of reflux esophago-laryngitis, modifying our previously reported model of chronic acid reflux esophagitis. METHODS: The modified chronic acid reflux esophagitis (m-RE) group (n = 10), in which the duodenum was wrapped with 2.5 mm of Nelaton catheter, was not treated with any drugs. Also postoperatively, two treatment groups (n = 10 in each) received different dosages of rabeprazole (RPZ): 1.0 mg/kg/day (RPZ 1.0 group) or 10.0 mg/kg/day (RPZ 10.0 group). As a control group (n = 5), other rats underwent sham operation. The esophagus and larynx were resected on day 14 after the operation, and ulcer score of the esophagus was assessed. The epithelial thickness and leukocyte infiltration of the supraglottic and subglottic laryngeal mucosae were investigated. The number of interleukin (IL)-1ß-positive cells was also counted and defined as the IL-1ß labeling index. RESULTS: In the m-RE group, the epithelial thickness, leukocyte infiltration, and IL-1ß labeling index of the supraglottic and subglottic laryngeal mucosae were increased compared with controls (P < 0.01). In the RPZ groups, not only the ulcer score of esophagus but also the epithelial thickness, leukocyte infiltration, and IL-1ß labeling index of both the supraglottic and subglottic laryngeal mucosae were decreased dose-dependently relative to the m-RE group (P < 0.05). CONCLUSIONS: Our modified chronic acid reflux esophagitis model proved useful in establishing a rat reflux esophago-laryngitis model, with both pathological laryngeal findings and reflux esophagitis shown to be improved by administration of a proton pump inhibitor.

Metastatic renal cell carcinoma diagnosed by capsule endoscopy and double balloon endoscopy.

BACKGROUND: Renal cell carcinoma commonly metastasizes to lung, liver, and bone. Small intestinal metastases are exceedingly rare. CASE REPORT: A 75-year-old man presented at our hospital with tarry stools. He had undergone a right nephrectomy for renal cell carcinoma (RCC) 6 years previously; in addition, he had received antiplatelet treatment for ischemic heart disease. Esophagogastroduodenoscopy, total colonoscopy, and computed tomography did not identify any cause for the gastrointestinal bleeding. He underwent capsule endoscopy (CE), which revealed an ulcerated submucosal tumor in the jejunum. We performed a double-balloon endoscopy (DBE), and histological findings identified a clear cell carcinoma. We diagnosed metastasis from the RCC. We performed a jejunectomy to resect the tumor and thus eliminate the source of the bleeding. CONCLUSIONS: CE and DBE are useful diagnostic tools. We recommend investigating the possibility of small intestinal metastases in cases of intestinal bleeding or anemia in patients with a history of malignant tumor.

Observational comparative trial of the efficacy of proton pump inhibitors versus histamine-2 receptor antagonists for uninvestigated dyspepsia.

BACKGROUND AND AIMS: It is still controversial which drugs, proton pump inhibitors (PPI) or histamine-2 receptor antagonists (H2RA), are more effective for dyspepsia in the Japanese population. METHODS: Patients with uninvestigated dyspepsia (n = 104; male/female 41/63) were treated with either rabeprazole 10 mg o.d. (n = 62) or lafutidine 10 mg b.i.d. (n = 42) for 4 weeks. Questionnaires (modified Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease [mFSSG] and quality of life [QOL], SF-8) were administered before and after therapy. The mFSSG was classified into a total score (Q-T), reflux score (Q-R), dyspepsia score (Q-D) and pain score (Q-P). The SF-8 had a physical component summary (PCS) and mental component summary (MCS). The predominant type of symptom was reflux (R-S), pain (P-S) or dysmotility (D-S). RESULTS: R-S was 19.2%, P-S 48.1%, D-S 24.0% and overlap 8.7%. In the R-S, Q-T and Q-R significantly improved with rabeprazole, but neither scale improved with lafutidine. MCS significantly improved with rabeprazole. In P-S, Q-T, Q-R, Q-D and Q-P significantly improved with both drugs. PCS significantly improved with both, whereas the MCS significant improved with rabeprazole. In D-S, Q-R and Q-D significant improved with rabeprazole, but neither improved with lafutidine. QOL did not improve with either. With overlap, neither scale nor the QOL reached a significant difference. CONCLUSION: Both PPI and H2RA have a positive effect on P-S, but H(2)RA therapy is limited for R-S and D-S, whereas PPI therapy is generally effective. Therefore, careful prescription based on symptoms is important.

Clinicopathological characteristics of colorectal cancer less than 10 mm in diameter and invading submucosa and below.

BACKGROUND AND AIMS: Identifying the invasive depth of cancers less than 10 mm in diameter remains a challenge. This study examines the clinicopathological characteristics of colorectal cancers less than 10 mm in diameter and invading submucosal layer (SM)3 and below, which require surgery and must never be treated by endoscopic mucosal resection. METHODS: We studied 54 cases of colorectal cancer less than 10 mm in diameter and invading the submucosa and deeper tissues, by dividing them into two groups: those invading SM1 and SM2 versus those invading SM3 and below. We investigated the clinicopathological characteristics of cancers invading SM3 and below by comparing them with cancers invading SM1 and SM2. Similarly, 38 cases, whose endoscopic findings could be analyzed, were selected and examined. RESULTS: In cases invading SM3 and below, the rates of moderately to poorly differentiated adenocarcinoma, lymphatic and venous permeation and lymph node metastasis were significantly higher than those invading SM1 and SM2. Among cases invading SM3 and below, the presence of endoscopic findings-including white spots of the protruded type, and fullness, white spots, hardness and protruded lesions in the depressed area of the depressed type-was significantly higher than among those invading SM1 and SM2. CONCLUSION: Colorectal cancers less than 10 mm in diameter and invading SM3 and below have high malignant potential. Cancers of this invasive depth can be identified by looking for characteristics such as white spots, fullness, hardness and protruded lesions in the depressed area. Careful endoscopic observation for these signs aids in determining the appropriate treatment.

Colonic mucosal lesions associated with long-term or short-term administration of nonsteroidal anti-inflammatory drugs.

AIM: The effects of short- or long-term administration of nonsteroidal anti-inflammatory drugs (NSAIDs) on the colon have not been well characterized. We assessed the risk of developing colonic mucosal lesions according to the duration of exposure to NSAIDs: short-term and/or long-term use. METHOD: A case-controlled study was performed by reviewing medical records for endoscopic findings, underlying disease, pre-endoscopic symptoms, category of NSAIDs used and duration of use. The patients underwent colonoscopy between January and October 2004, and 75 colitis cases and 1801 non-colitis controls were identified. The prevalence of NSAID use was compared between the cases and controls. The age- and sex- adjusted odds ratios (OR) were estimated using multiple logistic regression models. RESULTS: NSAIDs had been used in colitis cases and non-colitis controls for over six months in 20.0% and 12.7%, and for one week in 4.0% and 2.1%. Overall 76.0% and 85.2% had not received NSAIDs. The adjusted OR (95% confidence interval) for colonic mucosal lesions with short- and long term NSAID administration combined vs. non-use was 2.04 (1.16-3.61). When determined separately for short- and long-term NSAID users, the adjusted ORs were 1.48 (0.42-5.25) and 2.21 (1.19-4.11), compared to non-users. These values signify a trend toward an increased frequency of colonic mucosal lesions with longer use of NSAIDs (P=0.011 for trend). CONCLUSION: Long-term use of NSAIDs increased the risk of colonic mucosal lesions, suggesting that NSAIDs may contribute to the pathogenesis of colonic ulcer or colitis.

Endoscopic submucosal dissection of large colorectal tumors by using a novel spring-action S-O clip for traction (with video).

BACKGROUND: Endoscopic submucosal dissection (ESD) allows en bloc resection of large GI neoplasms, regardless of their size; however, technical difficulties associated with ESD in the colorectum make it less widely applied in the treatment of tumors in this region. To address this difficulty, we designed a rubber strip-based traction device, called the S-O clip (Sakamoto-Osada clip) and reported previously that ESD with this device was effective for complete resection of large, superficial colorectal neoplasms. In this report, we describe a novel spring-action version of the S-O clip (spring S-O clip) that improves the facility of clip use during ESD of colorectal tumors. OBJECTIVE: To evaluate the efficacy and safety of the spring S-O clip for ESD of colorectal neoplasms. DESIGN: Case series. SETTING: Juntendo University Hospital. MAIN OUTCOME MEASUREMENTS: The efficacy and safety of the spring S-O clip traction device during ESD of colorectal tumors. RESULTS: In 3 cases, a large, superficial neoplasm in the right side of the colon was removed safely and successfully en bloc without complication. Procedure times for the 3 cases were 44, 27, and 49 minutes, with resected specimens measuring 40, 24, and 35 mm, respectively. LIMITATION: Uncontrolled study. CONCLUSION: This limited case series demonstrates that spring S-O clip-assisted ESD is safe and effective for en bloc resection of large superficial neoplasms in the right side of the colon.

Attenuation of gastric mucosal inflammation induced by indomethacin through activation of the A2A adenosine receptor in rats.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin induce gastric mucosal lesions in part by the activation of inflammatory cells and the production of proinflammatory cytokines. The activation of adenosine A(2A) receptors inhibits inflammation by increasing cyclic AMP in leukocytes and reducing both the production of various proinflammatory cytokines and neutrophil chemotaxis. The aim of present study was to determine whether administration of an orally active adenosine A(2A) receptor agonist (4-[3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-piperidine-1-carboxylic acid methyl ester; ATL-313) ameliorated indomethacin-induced gastric mucosal lesions in rats. METHODS: Gastric lesions were produced by oral gavage of indomethacin (30 mg/kg). ATL-313 (1-10 microg/kg) was given orally just before the indomethacin administration. RESULTS: The ulcer index induced by indomethacin was significantly (>50%) reduced by pretreatment with ATL-313 and this effect was blocked completely by the addition of equimolar ZM241385, a selective A(2A) receptor antagonist. The gastric content of myeloperoxidase (MPO) and proinflammatory cytokines was significantly reduced by 10 microg/kg ATL-313, but gastric mucosal prostaglandin 2 (PGE2) was not affected. CONCLUSION: We conclude that ATL-313 does not inhibit the mucosal damaging effect of indomethacin, but it does block secondary injury due to stomach inflammation. A(2A) agonists may represent a class of new therapeutic drugs for NSAID-induced gastric ulcers.

Heat-shock protein 70-overexpressing gastric epithelial cells are resistant to indomethacin-induced apoptosis.

BACKGROUND/AIMS: Protecting intestinal mucosa from nonsteroidal anti-inflammatory drugs is still an unsolved problem. It has been revealed that apoptosis in epithelial cells as a result of mitochondrial injury is an important pathogenesis in indomethacin-induced gastric mucosal injury. In this study, we revealed the effect of overexpressed heat-shock protein 70 (HSP70) in indomethacin-induced apoptosis and oxidative stress. METHODS: HSP70-overexpressing rat gastric mucosal cells (7018-RGM-1 cells) and control cells (pBK-CMV-12 cells) were used and treated with 0-500 microM of indomethacin for 24 h. Cell viability and cytotoxity were measured by a WST-8 assay and a lactate dehydrogenase release assay, respectively. Apoptosis was observed by fluorescence microscopy staining with Hoechst 33342 and propidium iodide. The expression of Bcl-2 family proteins, activation of caspase-3, and 4-hydroxy-2-nonenal (4-HNE)-modified proteins were assessed by Western blot analysis. RESULTS: Indomethacin caused apoptosis of gastric epithelial cells. The 7018-RGM-1 cells survived significantly after indomethacin treatment compared to the control cells. The increase in pro-apoptotic Bad proteins, the decrease in anti-apoptotic Bcl-2 proteins, and caspase activation were all suppressed in the 7018-RGM-1 cells. A lower level of indomethacin-induced 4-HNE-modification was detected in the 7018-RGM-1 cells than in the control cells. CONCLUSION: Overexpressed HSP70 may potentiate resistance to apoptosis and oxidative stress in indomethacin-induced gastric epithelial cell injury.

Expression and function of stress (heat shock) proteins in gastrointestinal tract.

This review will focus on gastrointestinal mucosal protection against cytotoxic agents and cellular stress mainly from the viewpoint of expression and function of heat shock proteins, in their role of 'molecular chaperones', as internal cytoprotectants. Also, recently identified target molecules of heat shock protein in damaged gastric mucosal cells are introduced. Elucidation of such stress-responses and repairing process of damaged protein by heat shock proteins in the gastrointestinal mucosa may provide a better understanding for the mechanisms of cytoprotection and cellular repair. In addition, these findings in post-genomic level may provide new strategies for the therapy of gastrointestinal disorders.

Gastrointestinal tuberculosis as a cause of massive bleeding.

BACKGROUND: Although minor bleeding is fairly common among cases of gastrointestinal tuberculosis, massive hemorrhage necessitating blood transfusion is rare. CASE REPORT: A 72-year-old woman presented to the hospital with hematochezia. Colonoscopy revealed multiple ulcers, including round ulcers, throughout the large intestine. Upper gastrointestinal endoscopy revealed multiple duodenal ulcers. Hemostasis was performed on the bleeding point, but other untreated ulcers exhibited recurrent bleeding. Fecal culture results established the diagnosis of gastrointestinal tuberculosis. The patient was put on a standard antituberculosis regimen and remains in remission. CONCLUSIONS: Gastrointestinal tuberculosis should be considered as one of the causes for massive and intractable gastrointestinal bleeding.

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90.

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.

Double-balloon endoscopy for treatment of long-term abdominal discomfort due to small bowel penetration by an eel bone.

BACKGROUND: Penetration of the GI tract by a fish bone was treated by laparotomy. DBE is a useful procedure for removal of a foreign body from the small bowel. CASE REPORT: A 33-year-old male presented with bothersome postprandial fullness. He was diagnosed previously with functional dyspepsia, and had been treated with medication for 8 months with no success. During antegrade DBE, which was performed to rule out of small intestinal abnormalities, an eel bone found stuck in the jejunum was grasped with forceps and pulled out. Following removal of the bone, the patient's postprandial fullness ameliorated dramatically. CONCLUSIONS: This is the first report of DBE-based diagnosis and treatment of a small bowel penetration by a fish bone.

Gene expression profiling following constitutive activation of MEK1 and transformation of rat intestinal epithelial cells.

BACKGROUND: Constitutive activation of MEK1 (caMEK) can induce the oncogenic transformation of normal intestinal epithelial cells. To define the genetic changes that occur during this process, we used oligonucleotide microarrays to determine which genes are regulated following the constitutive activation of MEK in normal intestinal epithelial cells. RESULTS: Microarray analysis was performed using Affymetrix GeneChip and total RNA from doxycycline inducible RIEtiCAMEK cells in the presence or absence of doxycycline. MEK-activation induced at least a three-fold difference in 115 gene transcripts (75 transcripts were up-regulated, and 40 transcripts were down-regulated). To verify whether these mRNAs are indeed regulated by the constitutive activation of MEK, RT-PCR analysis was performed using the samples from caMEK expressing RIE cells (RIEcCAMEK cells) as well as RIEtiCAMEK cells. The altered expression level of 69 gene transcripts was confirmed. Sixty-one of the differentially expressed genes have previously been implicated in cellular transformation or tumorogenesis. For the remaining 8 genes (or their human homolog), RT-PCR analysis was performed on RNA from human colon cancer cell lines and matched normal and tumor colon cancer tissues from human patients, revealing three novel targets (rat brain serine protease2, AMP deaminase 3, and cartilage link protein 1). CONCLUSION: Following MEK-activation, many tumor-associated genes were found to have significantly altered expression levels. However, we identified three genes that were differentially expressed in caMEK cells and human colorectal cancers, which have not been previously linked to cellular transformation or tumorogenesis.