Mitsugu Sekimoto

Reprogramming of Mouse and Human Cells to Pluripotency Using Mature MicroRNAs

Induced pluripotent stem cells (iPSCs) can be generated from differentiated human and mouse somatic cells using transcription factors such as Oct4, Sox2, Klf4, and c-Myc. It is possible to augment the reprogramming process with chemical compounds, but issues related to low reprogramming efficiencies and, with a number of protocols, residual vector sequences, remain to be resolved. We show here that it is possible to reprogram mouse and human cells to pluripotency by direct transfection of mature double-stranded microRNAs (miRNAs). Our approaches use a combination of mir-200c plus mir-302 s and mir-369 s family miRNAs. Because this reprogramming method does not require vector-based gene transfer, it holds significant potential for biomedical research and regenerative medicine.

Stromal Myofibroblasts Predict Disease Recurrence for Colorectal Cancer

Purpose: Myofibroblasts, which are specifically differentiated fibroblasts, are thought to play a central role in the desmoplastic reaction, a dynamic stromal change closely associated with cancer development. Although fundamental studies suggest that myofibroblasts may either facilitate or inhibit cancer progression, cumulative evidence supports their role in promoting tumor progression. The aim of this study was to assess the value of myofibroblasts in the cancer stroma as an indicator of disease recurrence after colorectal cancer surgery. Experimental Design: Using computer-assisted image analysis, we quantified myofibroblasts in the cancer-associated stroma of 192 colorectal cancers using α-smooth muscle actin as a marker. Results: The cancer-associated stroma contained various numbers of myofibroblasts (0.35-19.0%; mean, 5.55 ± 3.85%). Tumors with abundant myofibroblasts were associated with shorter disease-free survival rate (P = 0.001) for stage II and III colorectal cancer. Multivariate analysis indicated that α-smooth muscle actin was a significant prognostic factor comparable with lymph node metastasis and superior to other tumor and stromal components, including histology of the tumor invasive front, peritumoral lymphocytic infiltration, and Crohns-like lymphoid reaction. Moreover, colorectal cancers with synchronous liver metastasis generally displayed an active desmoplastic reaction, which was retained in the metastatic lesion to a similar extent. Conclusions: The results suggest that the abundance of myofibroblasts in cancer-associated stroma may be a useful indicator of disease recurrence after curative colorectal cancer surgery.

Defined factors induce reprogramming of gastrointestinal cancer cells

Although cancer is a disease with genetic and epigenetic origins, the possible effects of reprogramming by defined factors remain to be fully understood. We studied the effects of the induction or inhibition of cancer-related genes and immature status-related genes whose alterations have been reported in gastrointestinal cancer cells. Retroviral-mediated introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins, including Nanog, Ssea4, Tra-1-60, and Tra-1-80 in esophageal, stomach, colorectal, liver, pancreatic, and cholangiocellular cancer cells. Induced cells, but not parental cells, possessed the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, which was supported by epigenetic studies, indicating methylation of DNA strands and the histone H3 protein at lysine 4 in promoter regions of pluripotency-associated genes such as NANOG. In in vitro analysis induced cells showed slow proliferation and were sensitized to differentiation-inducing treatment, and in vivo tumorigenesis was reduced in NOD/SCID mice. This study demonstrated that pluripotency was manifested in induced cells, and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation-inducing treatment. Retroviral-mediated introduction of iPC cells confers higher sensitivity to chemotherapeutic agents and differentiation-inducing treatment.

Extraction and Analysis of Diagnostically Useful Proteins from Formalin-fixed, Paraffin-embedded Tissue Sections

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-μm-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as β-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

High Incidence of Thrombosis of the Portal Venous System After Laparoscopic Splenectomy: A Prospective Study With Contrast-Enhanced CT Scan

Objective:The aims of this prospective study were to investigate the true incidence of portal or splenic vein thrombosis (PSVT) after elective laparoscopic splenectomy using contrast-enhanced computed tomography (CT) scan, and outcome of anticoagulant therapy for PSVT. Summary Background Data:Although rare, thrombosis of the portal venous system is considered a possible cause of death after splenectomy. The reported incidence of ultrasonographically detected PSVT after elective open splenectomy ranges from 6.3% to 10%. Methods:Twenty-two patients underwent laparoscopic splenectomy (LS group), and 21 patients underwent open splenectomy (OS group). Preoperative and postoperative helical CT with contrast were obtained in all patients, and the extent of thrombosis was investigated. Prothrombotic disorder was also determined. Results:PSVT occurred in 12 (55%) patients of the LS group, but in only 4 (19%) of the OS group. The difference was significant (P = 0.03). Clinical symptoms appeared in 4 of the 12 LS patients. Thrombosis occurred in the intrahepatic portal vein (n = 9), extrahepatic portal vein (n = 2), mesenteric veins (n = 1), proximal splenic vein (n = 4), and distal splenic vein (n = 8). Prothrombotic disorder was diagnosed in 1 patient. Anticoagulant therapy was initiated once the diagnosis was established, and complete recanalization, except for distal splenic vein, was observed without any adverse event. Patients with splenomegaly were at high risk of PSVT. Conclusions:PSVT is a more frequent complication of laparoscopic splenectomy than previously reported but can be treated safely following early detection by CT with contrast.

Comparative Detection of Lymph Node Micrometastases of Stage II Colorectal Cancer by Reverse Transcriptase Polymerase Chain Reaction and Immunohistochemistry

PURPOSE Inconsistent conclusions have been drawn about the clinical significance of micrometastases in lymph nodes (LNs) of node-negative colorectal cancer (CRC) patients. We performed a comparative study of detection of micrometastases using immunohistochemistry (IHC) by anti-cytokeratin antibody and carcinoembryonic antigen (CEA)-specific reverse-transcriptase polymerase chain reaction (RT-PCR) in the same patients, in an attempt to move closer to their clinical application. PATIENTS AND METHODS Sixty-four CRC patients, with RNA of good quality available from paraffin-embedded LN specimens, were selected from 84 stage II patients who underwent curative surgery between 1988 and 1996. We investigated associations between the presence of micrometastases by each method and prognosis. RESULTS Micrometastases were detected in 19 (29.6%) of 64 patients by RT-PCR and in 35 (54.7%) of 64 patients by IHC. By RT-PCR analysis, patients exhibiting a positive band for CEA mRNA had a significantly worse prognosis than those who were RT-PCR-negative, with respect to both disease-free and overall survival (P =.027 and.015, respectively). By IHC analysis, the presence of micrometastasis did not predict patient outcome in terms of either disease-free or overall survival. Infiltrating pattern of tumor growth characteristic was significantly associated with shorter disease-free survival among various clinical or pathologic factors. By multivariate Cox regression analysis, micrometastasis detected by RT-PCR and the Crohns-like lymphoid reaction were both independent prognostic factors. CONCLUSION Micrometastases detected by RT-PCR, but not IHC, may be of clinical value in identifying patients who may be at high risk for recurrence of CRC and who are therefore likely to benefit from systemic adjuvant therapy.

JTE-522, a cyclooxygenase-2 inhibitor, is an effective chemopreventive agent against rat experimental liver fibrosis1 ☆

BACKGROUND & AIMS The aim of this study was to assess the effects of cyclooxygenase (COX)-2 inhibition on rat experimental liver fibrogenesis. METHODS We investigated the inhibitory effects of a selective COX-2 inhibitor, JTE-522, on liver fibrosis induced by a choline-deficient, l-amino acid-defined diet (CDAA). Inhibitory effect was also tested in a second model of thioacetamide (TAA)-induced liver fibrosis. RESULTS CDAA induced liver fibrosis and preneoplastic foci at 12 weeks and cirrhosis at 36 weeks. Hepatocellular carcinoma was noted in 13 of 15 rats (87%). JTE-522 significantly inhibited fibrosis and development of preneoplastic lesions in a dose-dependent manner and completely inhibited generation of cirrhosis and hepatocellular carcinoma at both low and high doses (10 and 30 mg/kg body wt/day, respectively). JTE-522 administrated only from 12 weeks to 36 weeks also prevented cirrhosis and formation of hepatocellular carcinoma. JTE-522 itself did not cause local or systemic gross or histopathologic changes at 36 weeks. Mechanistic studies indicated that the CDAA model displayed up-regulation of several biomarkers, including COX-2, arachidonate metabolite (prostaglandin E(2)), serum aspartate aminotransferase, and c-myc expression. The model also showed an increased proportion of activated hepatic stellate cells, proliferating cell nuclear antigen index, and CD45-positive inflammatory cells in the liver. JTE-522 effectively diminished these changes. JTE-522 exhibited similar antifibrosis effects in the TAA model. CONCLUSIONS Our results suggest that COX-2 is involved in CDAA- and TAA-induced liver fibrosis. Our data also indicate that JTE-522 is a potent chemopreventive agent of rat liver fibrosis with low toxicity.

Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection

BackgroundThere are currently no accurate serum markers for detecting early risk of colorectal cancer (CRC). We therefore developed a non-targeted metabolomics technology to analyse the serum of pre-treatment CRC patients in order to discover putative metabolic markers associated with CRC. Using tandem-mass spectrometry (MS/MS) high throughput MS technology we evaluated the utility of selected markers and this technology for discriminating between CRC and healthy subjects.MethodsBiomarker discovery was performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Comprehensive metabolic profiles of CRC patients and controls from three independent populations from different continents (USA and Japan; total n = 222) were obtained and the best inter-study biomarkers determined. The structural characterization of these and related markers was performed using liquid chromatography (LC) MS/MS and nuclear magnetic resonance technologies. Clinical utility evaluations were performed using a targeted high-throughput triple-quadrupole multiple reaction monitoring (TQ-MRM) method for three biomarkers in two further independent populations from the USA and Japan (total n = 220).ResultsComprehensive metabolomic analyses revealed significantly reduced levels of 28-36 carbon-containing hydroxylated polyunsaturated ultra long-chain fatty-acids in all three independent cohorts of CRC patient samples relative to controls. Structure elucidation studies on the C28 molecules revealed two families harbouring specifically two or three hydroxyl substitutions and varying degrees of unsaturation. The TQ-MRM method successfully validated the FTICR-MS results in two further independent studies. In total, biomarkers in five independent populations across two continental regions were evaluated (three populations by FTICR-MS and two by TQ-MRM). The resultant receiver-operator characteristic curve AUCs ranged from 0.85 to 0.98 (average = 0.91 ± 0.04).ConclusionsA novel comprehensive metabolomics technology was used to identify a systemic metabolic dysregulation comprising previously unknown hydroxylated polyunsaturated ultra-long chain fatty acid metabolites in CRC patients. These metabolites are easily measurable in serum and a decrease in their concentration appears to be highly sensitive and specific for the presence of CRC, regardless of ethnic or geographic background. The measurement of these metabolites may represent an additional tool for the early detection and screening of CRC.

Quantitative evaluation of vimentin expression in tumour stroma of colorectal cancer

Recent studies have identified vimentin, a type III intermediate filament, among genes differentially expressed in tumours with more invasive features, suggesting an association between vimentin and tumour progression. The aim of this study, was to investigate whether vimentin expression in colon cancer tissue is of clinical relevance. We performed immunostaining in 142 colorectal cancer (CRC) samples and quantified the amount of vimentin expression using computer-assisted image analysis. Vimentin expression in the tumour stroma of CRC was associated with shorter survival. Overall survival in the high vimentin expression group was 71.2% compared with 90.4% in the low-expression group (P=0.002), whereas disease-free survival for the high-expression group was 62.7% compared with 86.7% for the low-expression group (P=0.001). Furthermore, the prognostic power of vimentin for disease recurrence was maintained in both stage II and III CRC. Multivariate analysis suggested that vimentin was a better prognostic indicator for disease recurrence (risk ratio=3.5) than the widely used lymph node status (risk ratio=2.2). Vimentin expression in the tumour stroma may reflect a higher malignant potential of the tumour and may be a useful predictive marker for disease recurrence in CRC patients.

Correlation of Beta-Catenin and Cyclin D1 Expression in Colon Cancers

Objective: Beta-catenin activates transcription by TCF/LEF and has been regarded as an oncogene in a wide range of malignant tumors. Among various molecules regulated by beta-catenin/Tcf, cyclin D1 is the most likely candidate for stimulation of the oncogenic pathway. The association between beta-catenin and cyclin D1 was investigated using clinical samples from colorectal cancers. Methods: The expression of beta-catenin and cyclin D1 was investigated by immunohistochemical analyses of samples from 70 patients with colorectal cancers. In 28 of the fresh tumor samples, beta-catenin protein was separated into soluble and insoluble fractions and quantitatively correlated with cyclin D1 protein by Western blot analysis. Results: Compared with noncancerous epithelium, beta-catenin and cyclin D1 were overexpressed (+) in 35 (50%) and 30 cases (43%), respectively. Cyclin D1 (+) was observed in 74% (26/35) of beta-catenin (+) cases, but only in 11% (4/35) of the beta-catenin (–) cases. Thus, there was a strong association between the expression of beta-catenin and that of cyclin D1 (p < 0.001). In the Western blot analysis, the amount of cyclin D1 correlated well with beta-catenin expression in the soluble fraction (p = 0.0016), but not with beta-catenin in the insoluble fraction or with E-cadherin expression. Beta-catenin (–)/cyclin D1 (–) cases displayed less tumor invasion than the remaining cases. However, there were no significant differences in lymph node metastasis or other clinicopathological findings. Conclusion: Our results indicate that beta-catenin overexpression in the cytoplasm may promote malignant transformation by triggering cyclin D1 expression in colorectal cancers.