Maintenance of Viability and Function of Rat Islets With the Use of ROCK Inhibitor Y-27632.

The number of patients with diabetes is on an increasing trend, thus leading to the belief that diabetes will be the largest medical problem of the 21st century. Islet transplantation can improve glycometabolic control in patients with type 1 diabetes. We studied the viability of Rho-associated protein kinase (ROCK) inhibitor Y-27632 in a culture system in vitro on freshly isolated rat islets. Islet isolation was conducted on a Lewis rat, and studies of culture solutions were split into two groups, one group using ROCK inhibitor Y-27632, and another without. On the seventh day of culture, we evaluated the differences for the cell morphology, viability, and insulin secretion. The Y-27632 group maintained form better than the group without Y-27632. With strong expression of Bcl-2 observed with the Y-27632 group, and expression suppressed with Bax, inhibition of apoptosis by Y-27632 was confirmed. The Y-27632 group predominantly secreted insulin. For islet transplantation, Y-27632 inhibited cell apoptosis in a graft and was also effective in promoting insulin secretion. We were able to confirm effective morphological and functional culture maintenance by separating islets from a rat and adding ROCK inhibitor Y-27632 to the medium.

Cryopreservation of rat islets of Langerhans by vitrification.

Cryopreservation could be a possible means of addressing the shortage of islets of Langerhans. We investigated the effects of EDT324 solution on the vitrification of isolated rat islets of Langerhans. Rat pancreatic islets were cryopreserved in 10% DMSO by a slow-rate freezing method or were cryopreserved in EDT324 solution by vitrification. The cryopreserved islets were compared in terms of viability, stimulation index and metabolic function after transplantation. After cryopreservation, the viability and stimulation of islets stored in EDT324 were 92.4% and 6.4, respectively, and were higher than islets stored by slow freezing (72.5% and 1.5, respectively). Streptozotocin-induced diabetic rats were transplanted with islets cryopreserved in EDT324, which corrected diabetes and achieved euglycemia within 2 days after transplantation. These results indicate that EDT324 allows successful cryopreservation of rat islets for long-term storage as an alternative solution to traditionally used solutions, such as 10% DMSO. Transplantation of cryopreserved islets into diabetic rats can achieve euglycemia.

Establishment of an immortalized porcine liver cell line JSNK-1 with retroviral transduction of SV40T.

Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1α1, collagen type 1α2, FLT-1, ß-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and α-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.

Characteristics of CD133(+) human colon cancer SW620 cells.

Worldwide, colorectal cancer is the third most common type of cancer affecting both sexes. It has been proposed that a small subset of cancer cells (cancer stem cells) within each tumor is able to initiate tumor growth. In 2007, two research groups simultaneously identified a colon cancer stem cell population in human tumors by the use of CD133 expression. In the present study, we used a human colon cancer cell line, SW620, to analyze the cancer stem cell-like characteristics of CD133(+) cells in vitro and in vivo. In vitro, CD133(+) SW620 cells had a higher proliferative capacity, were more irradiation- and chemotherapy-resistant, and had a higher expression of ß-catenin compared with CD133(-) cells. Injections of either CD133(+) or CD133(-) cells into the skin or rectal mucosa of NOD/SCID mice led to tumors; however, injection of CD133(+) cells resulted in the formation of larger tumors. Tumors derived from injections of CD133(-) cells did not contain any CD133(+) cells, whereas tumors derived from injections of CD133(+) cells did contain CD133(+) cells, suggesting self-renewing capability. However, the proportion of CD133(+) cells in the newly formed tumors in vivo was lower than the proportion of CD133(+) cells in vitro. In conclusion, the human colon cancer cell line, SW620, contains both CD133(+) and CD133(-) phenotypes, and the CD133(+) phenotype has characteristics consistent with those of cancer stem cells.

Analysis of fecal DNA methylation to detect gastrointestinal neoplasia.

BACKGROUND: The development of noninvasive screening tests is important to reduce mortality from gastrointestinal neoplasia. We sought to develop such a test by analysis of DNA methylation from exfoliated cancer cells in feces. METHODS: We first analyzed methylation of the RASSF2 and SFRP2 gene promoters from 788 primary gastric and colorectal tissue specimens to determine whether methylation patterns could act as stage-dependent biomarkers of gastrointestinal tumorigenesis. Next, we developed a novel strategy that uses single-step modification of DNA with sodium bisulfite and fluorescence polymerase chain reaction methodology to measure aberrant methylation in fecal DNA. Methylation of the RASSF2 and SFRP2 promoters was analyzed in 296 fecal samples obtained from a variety of patients, including 21 with gastric tumors, 152 with colorectal tumors, and 10 with non-neoplastic or inflammatory lesions in the gastrointestinal lumen. RESULTS: Analysis of DNA from tissues showed presence of extensive methylation in both gene promoters exclusively in advanced gastric and colorectal tumors. The assay successfully identified one or more methylated markers in fecal DNA from 57.1% of patients with gastric cancer, 75.0% of patients with colorectal cancer, and 44.4% of patients with advanced colorectal adenomas, but only 10.6% of subjects without neoplastic or active diseases (difference, gastric cancer vs undiseased = 46.5%, 95% confidence interval (CI) = 24.6% to 68.4%, P < .001; difference, colorectal cancer vs undiseased = 64.4%, 95% CI = 53.5% to 75.2%, P < .001; difference, colorectal adenoma vs undiseased = 33.8%, 95% CI = 14.2% to 53.4%, P < .001). CONCLUSIONS: Methylation of the RASSF2 and SFRP2 promoters in fecal DNA is associated with the presence of gastrointestinal tumors relative to non-neoplastic conditions. Our novel fecal DNA methylation assay provides a possible means to noninvasively screen not only for colorectal tumors but also for gastric tumors.

Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer.

BACKGROUND & AIMS: Colorectal cancers (CRCs) with the CpG island methylator phenotype (CIMP) often associate with epigenetic silencing of hMLH1 and an activating mutation in the BRAF gene. However, the current CIMP criteria are ambiguous and often result in an underestimation of CIMP frequencies in CRCs. Because BRAF and KRAS belong to same signaling pathway, we hypothesized that not only mutations in BRAF but mutant KRAS may also associate with CIMP in CRC. METHODS: We determined the methylation status in a panel of 14 markers (7 canonical CIMP-related loci and 7 new loci), microsatellite instability status, and BRAF/KRAS mutations in a collection of 487 colorectal tissues that included both sporadic and Lynch syndrome patients. RESULTS: Methylation analysis of 7 CIMP-related markers revealed that the mean number of methylated loci was highest in BRAF-mutated CRCs (3.6) vs KRAS-mutated (1.2, P < .0001) or BRAF/KRAS wild-type tumors (0.7, P < .0001). However, analyses with 7 additional markers showed that the mean number of methylated loci in BRAF mutant tumors (4.4) was the same as in KRAS mutant CRCs (4.3, P = .8610). Although sporadic microsatellite instability high tumors had the highest average number of methylated markers (8.4), surprisingly, Lynch syndrome CRCs also demonstrated frequent methylation (5.1). CONCLUSIONS: CIMP in CRC may result from activating mutations in either BRAF or KRAS, and the inclusion of additional methylation markers that correlate with mutant KRAS may help clarify CIMP in future studies. Additionally, aberrant DNA methylation is a common event not only in sporadic CRC but also in Lynch syndrome CRCs.

Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis.

O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous polyps (AP), 220 normal colonic mucosa from CRC patients (N-C) and 36 normal colonic mucosa specimens obtained from subjects without colorectal neoplasia (N-N) by combined bisulfite restriction analysis (COBRA). The region-specific methylation data were compared to the MGMT protein expression, spectrum of KRAS mutations and other clinical features. Extensive (including both Mp and Eh) and partial (either Mp or Eh) MGMT methylation were found in 24.5% and 11.6% of CRCs, 3.8% and 27.9% of APs, 0.5% and 7.7% of C-Ns and 2.8% and 2.8% of N-Ns, respectively. Extensive methylation of MGMT promoter was primarily present in CRCs while partial methylation was common in APs. Extensive methylation of MGMT promoter was associated with loss/reduced protein expression (p < 0.0001), as well as with G to A mutations in KRAS (p = 0.0017). We herein provide first evidence that extensive methylation of MGMT promoter region is essential for methylation-induced silencing of this gene. Our data suggest that MGMT methylation may evolve and spread throughout the promoter in a stepwise manner as the colonic epithelial cells progress through the classical-adenoma-cancer multistep cascade.

Microsatellite instability and clinicopathological features in esophageal squamous cell cancer.

Since multiple genetic alterations are involved in the molecular pathogenesis of esophageal squamous cell cancer (ESCC), the role of microsatellite instability (MSI) in its carcinogenesis is not well defined. The reported frequency of MSI in ESCC ranges from 2 to 66.7% but the majority of the results are derived from relatively small studies. Therefore, we carried out a precise MSI analysis on a large number of ESCC samples to clarify the significance of MSI in the ESCC tumorigenesis. The MSI status of the DNA extracted from 62 ESCC samples and 62 counterpart-normal esophageal epitheliums were studied with five NCI panel markers and ten microsatellite markers located in 17q24-25. Forty-four paraffin-embedded samples and 18 frozen samples from the ESCC patients who underwent surgery were studied. The MSI status was classified as MSS (microsatellite stable), MSI-L (low-level MSI; <30% of markers examined showed instability) and MSI-H (high-level MSI; >30% of markers reported instability). Among the 62 ESCC cases analyzed by the 15 microsatellite markers, 38 out of 62 cases (61.3%) showed MSS, 19 out of 62 cases (30.6%) showed MSI-L and 5 out of 62 cases (8.1%) showed MSI-H. Although the MSI status was not associated with the status of lymph node metastasis or a histological type of cancer, the depth of cancer invasion was significantly associated with the frequency of MSS status and the levels of MSI-L were inversely correlated with the depth of invasion (T1/T2 vs. T3/4; P=0.0007). However, MSI status was not associated with the prognosis of the ESCC patients. This is the first large scale MSI analysis of the ESCC in comparison with the clinicopathological features. Relatively high frequency of MSI-L was observed in ESCC and the frequency of MSI-L was inversely correlated with the depth of invasion.

Heterogeneous microsatellite instability observed within epithelium of ulcerative colitis.

Microsatellite instability (MSI) has been associated with colitic cancer. However, reported frequency of MSI was varied and the association of MSI with mismatch repair (MMR) deficiency was unclear. In addition, the occurrence of genetic alterations in stromal cells within ulcerative colitis (UC) is still controversial. We therefore sampled 164 microareas in various pathological lesions of UC with or without colitic cancer and studied the MSI status in relation to the DNA repair protein expressions. A total of 129 microfoci from colorectal tissue of 5 colitic cancer patients and 35 microfoci of 7 UC patients (without neoplasm) were carefully sampled by laser-capture microdissection. MSI was analyzed in each microsamples. The protein expression of MMR genes (MLH1, MSH2, MSH6), O(6)-methylguanine-DNA methyltransferase and p53 were assessed by immunohistochemical analysis. Variety of di-nulcleotide microsatellite markers was altered in individual microfoci from different morphological epithelial lesions, in full range of nonneoplastic epithelium to colitic cancer. Interestingly, MSI was not observed in stromal cells at any sites, including those within colitic cancer/dysplasia lesions. Expression of the MMR proteins was not lost in any of the lesions examined. Microsatellite alterations rather seem to be related to the initiation than to the progression of colitic cancer.

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients.

Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin-biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes.

Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation.

PURPOSE: BRAF mutations are common in sporadic colorectal cancers (CRCs) with a DNA mismatch repair (MMR) deficiency that results from promoter methylation of hMLH1, whereas KRAS mutations are common in MMR proficient CRCs associated with promoter methylation of MGMT. The aim of this study was to further investigate the link between genetic alterations in the RAS/RAF/ERK pathway and an underlying epigenetic disorder. PATIENTS AND METHODS: Activating mutations of BRAF and KRAS were identified and correlated with promoter methylation of 11 loci, including MINT1, MINT2, MINT31, CACNA1G, p16(INK4a), p14(ARF), COX2, DAPK, MGMT, and the two regions in hMLH1 in 468 CRCs and matched normal mucosa. RESULTS: BRAF V599E mutations were identified in 21 (9%) of 234 CRCs, and KRAS mutations were identified in 72 (31%) of 234 CRCs. Mutations in BRAF and KRAS were never found in the same tumor. CRCs with BRAF mutations showed high-level promoter methylation in multiple loci, with a mean number of methylated loci of 7.2 (95% CI, 6.6 to 7.9) among 11 loci examined (P < .0001). Tumors with KRAS mutations showed low-level promoter methylation, and CRCs with neither mutation showed a weak association with promoter methylation, with an average number of methylated loci of 1.8 (95% CI, 1.5 to 2.1) and 1.0 (95% CI, 0.79 to 1.3), respectively. CONCLUSION: In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.

Allele-specific methylation analysis on upstream promoter region of H19 by methylation-specific PCR with confronting two-pair primers.

H19 and IGF2 genes are imprinted genes and expressed differently depending on whether they are carried by a chromosome of maternal or paternal origin; H19 is expressed only from the maternal allele and IGF2 only from the paternally inherited allele. The upstream promoter region of H19 has the imprinting-control region (ICR) or CTCF binding sites, where the methylation status of this region is critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15. There are various reports on imprinting disorders in this region. In colorectal cancer aberrant biallelic methylation of CTCF binding site has been reported, and aberrant hypomethylation of this region in bladder cancer. Thus, certain human neoplasms have either hyper- or hypo-methylation in the ICR. Hence it is still difficult to analyze allele-specific methylation disorder of the region, or differentially methylated regions (DMR), locate upstream of H19. Here we report a new method, which could distinguish paternal epigenetic or maternal epigenetic pattern by a single PCR assay, to combine methylation-specific PCR and PCR with confronting two-pair primers (MSP-CTPP). Using this method, we investigated the region close to H19 ICR in 161 colorectal cancer and 65 gastric cancer cases.

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density.

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.

Allelic loss of a common microsatellite marker MYCL1: a useful prognostic factor of poor outcomes in colorectal cancer.

PURPOSE: Allelic loss involving chromosome arms 5q, 8p, 17p, and 18q is commonly detected in colorectal cancer (CRC). The short arm of chromosome 1 is also frequently affected in a whole range of cancer types, including CRC. Our aim in the present study was to determine whether allelic losses on 1p were likely to be of much value in predicting the prognosis of CRC cases. EXPERIMENTAL DESIGN: Genomic DNA was prepared from tumor and corresponding normal tissue specimens from 90 patients who had undergone curative resection for CRC. Loss of heterozygosity (LOH) on chromosome arms 1p, 2p, 5q, 7q, 8p, 17p, 17q, and 18q was examined using 14 microsatellite markers, and possible correlations between LOH and clinicopathological factors (including tumor recurrence and patient survival) were investigated. LOH at the MYCL1 microsatellite marker at 1p34 was detected in 12 of 74 (16.2%) patients who were informative for this marker. RESULTS: After controlling for tumor stage and gender and excluding findings for patients with remote metastasis, we found that patients who were positive for LOH at MYCL1 were 31 times more likely to experience recurrence than those who were negative for LOH at this locus (95% confidence intervals, 2.27- infinity; P = 0.04). There were indications of a similar tendency for LOH at the 14-3-3-sigma-TG microsatellite marker at 1p35, but we could find no evidence of a significant association between LOH at this site and tumor recurrence or patient survival. We were also unable to detect significant association between LOH at the various sites on 2p, 5q, 7q, 8p, 17p, 17q, and 18q and either tumor recurrence or patient survival. CONCLUSIONS: CRC patients whose tumors exhibited LOH at MYCL1 at chromosome 1p34 were likely to have a poor prognosis, suggesting that this marker may have clinical relevance.

Hypermethylation of O6-methylguanine-DNA methyltransferase promoter may predict nonrecurrence after chemotherapy in colorectal cancer cases.

PURPOSE: Because O(6)-methylguanine-DNA methyltransferase (MGMT) plays an essential role in repairing DNA damage caused by environmental alkylating chemicals, we were interested in determining whether we could see any obvious changes in the properties of colorectal cancers (CRCs) in which the MGMT gene had been silenced by hypermethylation and hence in which very few MGMT protein molecules were being produced. EXPERIMENTAL DESIGN: We used a methylation-specific PCR assay to determine the methylation status of the MGMT promoter in the DNA molecules extracted from CRC and nontumor tissue samples from 116 patients who had undergone CRC surgery and for whom clinical outcome information was available on file. RESULTS: We found evidence of MGMT promoter hypermethylation in 26 of 90 CRC cases, and we noted that the later the stage at which a tumor was diagnosed, the less likely its MGMT promoter was to be methylated (P = 0.03, adjusting for chemotherapy), especially for stage D patients (P = 0.01). We also found that CRC patients with unmethylated MGMT promoters were much more likely to experience recurrence within 36 months than patients with hypermethylated MGMT promoters (crude odds ratio, 14.0; 95% confidence interval, 2.42-81.01). After adjustment for stage, CRC patients with unmethylated MGMT promoters who had been exposed to chemotherapy were found to have a 5.3-fold greater risk of recurrence than those who had no exposure to chemotherapy (95% confidence interval, 1.15-30.92). CONCLUSIONS: Hypermethylation of the MGMT promoter may be predictive of a low risk of recurrence in CRC patients receiving adjuvant chemotherapy.