Gensaku Okada

Expression of mesothelin mRNA in pure pancreatic juice from patients with pancreatic carcinoma, intraductal papillary mucinous neoplasm of the pancreas, and chronic pancreatitis.

Objectives: In the gene expression analysis of pancreatic carcinoma (PCa) using serial analysis of gene expression (SAGE) according to Ryu et al, the tag for the mesothelin mRNA transcript was present in 7 of 8 SAGE libraries derived from PCa but not in the 2 SAGE libraries derived from normal pancreatic duct epithelial cells. Mesothelin mRNA expression was confirmed with in situ hybridization in all 4 resected primary PCa tumors and with RT-PCR in 18 of 20 PCa cell lines, whereas mesothelin protein expression was confirmed with immunohistochemistry in all 60 resected primary PCa tissues by Argani et al. We evaluated mesothelin mRNA expression in pure pancreatic juice (PPJ) obtained from patients with PCa, chronic pancreatitis (CP), and intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Methods: We evaluated mesothelin mRNA expression in the PPJ obtained from 21 patients with PCa, 22 with CP, and 11 with IPMN with reverse transcriptase PCR (RT-PCR). The PCR products were analyzed with agarose gel electrophoresis. DNase I treatment before RT-PCR and direct sequencing of the RT-PCR bands were performed for the analysis of the RT-PCR bands. Results: Two products, of 308 and 226 bp, were obtained with RT-PCR, and the 308-bp RT-PCR product was confirmed as being that derived from the genomic DNA by direct DNA sequencing. Mesothelin mRNA expression was discovered using RT-PCR in 11 (52%) of 21 patients with PCa, 5 (45%) of 11 with IPMN, and 3 (14%) of 22 with CP. Fishers exact test revealed significant differences between PCa and CP for mesothelin mRNA (P < 0.01). Moreover, the RT-PCR product (226 bp) of mesothelin mRNA in the PPJ samples with PCa was generally stronger than that in the PPJ samples with IPMN. Conclusion: Expression of mesothelin mRNA in PPJ was not strictly specific to PCa and was apt to be stronger in PCa than in IPMN. Quantitative detection of mesothelin mRNA in PPJ may have potential diagnostic implications for pancreatic tumors.

Diagnostic utility of aberrant methylation of tissue factor pathway inhibitor 2 in pure pancreatic juice for pancreatic carcinoma

The tissue factor pathway inhibitor 2 (TFPI‐2) is a Kunitz‐type serine proteinase inhibitor. Recently, the aberrant methylation of TFPI‐2 was detected frequently in pancreatic carcinoma (PCa) tissues but not in normal pancreatic tissues. We analyzed the aberrant methylation of TFPI‐2 in the pure pancreatic juice (PPJ) aspirated endoscopically from patients with various pancreatic diseases. Using the highly sensitive methylation‐specific polymerase chain reaction (MSP) and quantitative MSP (Q‐MSP) assay, we investigated the aberrant methylation of TFPI‐2 in nine human PCa cell lines and in the PPJ from patients with PCa, intraductal papillary mucinous neoplasms (IPMN) and chronic pancreatitis (CP). The incidence of aberrant TFPI‐2 methylation was seven (77.8%) of nine PCa cell lines by Q‐MSP. In cell lines, the expression of TFPI‐2 mRNA by quantitative reverse transcription–polymerase chain reaction showed an inverse correlation to the aberrant methylation of TFPI‐2. The incidence of aberrant TFPI‐2 methylation in the PPJ was 21 (58.3%) of 36 PCa patients, three (17.6%) of 17 IPMN and one (4.8%) of 21 CP by MSP assay. Using a suitable cut‐off value of 2.5 according to the receiver operating characteristic curve, the incidence of aberrant TFPI‐2 methylation in the PPJ by real‐time MSP was 18 (62.1%) of 29 PCa patients, one (5.1%) of 17 IPMN and three (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI‐2 hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (P < 0.001) or CP (P < 0.001). Moreover, the aberrant methylation rate of TFPI‐2 in the PPJ was 100%, as observed (6/6) in the PCa patients with liver metastasis, and 86.7% (26/30) in stages IVa + IVb of PCa by Q‐MSP assay. These results suggest that promoter methylation of TFPI‐2 in the PPJ may be a useful marker in the diagnosis and progression of PCa using an endoscopically feasible approach. (Cancer Sci 2006; 97: 1267–1273)

Preproenkephalin hypermethylation in the pure pancreatic juice compared with p53 mutation in the diagnosis of pancreatic carcinoma

BackgroundAberrant methylation of CpG islands is a common mechanism for the dysregulation of tumor suppressor genes in a variety of human malignancies. Preproenkephalin ppENK) hypermethylation is recognized in 90% of pancreatic carcinoma (PCa) tissues, but not in normal pancreas. We analyzed ppENK hypermethylation in pure pancreatic juice (PPJ) in patients with PCa, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis (CP), and elucidated its usefulness as a marker in the diagnosis of PCa compared with p53 mutation.MethodsPPJ was collected endoscopically from 28 patients with PCa, 15 patients with IPMN, and 20 patients with CP. DNA was extracted from the supernatant and the sediment of PPJ. Methylation-specific polymerase chain reaction was performed for hypermethylation analysis of ppENK. In addition, single-strand conformation polymorphism and direct sequencing were performed simultaneously to identify p53 mutations.ResultsThe incidence of ppENK hypermethylation in the supernatant and/or the sediment of PPJ was 50% (14 of 28) in patients with PCa. In contrast, the incidence of ppENK hypermethylation was 26.7% (4 of 15) in patients with IPMN, and 5% (1 of 20) in patients with CP (P < 0.002). The incidence of p53 mutations in the PPJ was 42.9% (12 of 28) in patients with PCa and 0% (0 of 20) in patients with CP. Furthermore, the incidence of ppENK hypermethylation and/or p53 mutations in the PPJ was enhanced to 67.9% (19 of 28) in patients with PCa in the combination assay.ConclusionsThese results suggest that ppENK hypermethylation in PPJ is specific for cancer, and the combination assay with p53 enhances the genetic diagnosis of PCa.

Establishment of a metastatic murine cell line carrying the human c-Ha-ras

Dear Editor: Development of new modalities to control metastasis is an urgent requirement of cancer therapy. However, the available methods have an inherent limitation in detecting and quantifying micro-metastases. It is possible to experimentally detect metastasis if genes of a species are detected in the tissues of another animal. Therefore, a new system needs to be established which consists of: 1) ceils that grow and metastasize in immunocompetent syngeneic animals, a n d 2) cells that have genes which are distinguishable from those of the animal tissues. The combination of r/mHM-SFME-1 cells and Balb/c mice provides a good model system for this application. The r/mHM-SFME1 cells are derived from ras/myc SFME cells transformed by activated human c-Ha-ras and mouse c-myc genes (5,7). Since original serum-free mouse embryo (SFME) cells were established from a Balb/c mouse embryo (3), immunocompetent Balb/c mice are syngeneic for both ras/myc SFME and r/mHM-SFME-1 cells. Here we describe the establishment of the r/mHM-SFME-1 cell line in vitro. One million of G418-resistant ras/myc SFME cells, which were transfected with pSV2-neo by calcium-phosphate co-precipitation (7), were injected subcutaneously into the backs of Balb/c mice. All of mice, which developed solid tumors within 2 months, were sacrificed to check metastases. One of them had metastases in the subaxillary and submaxillary lymph nodes, the lung and the liver. The metastases were excised from each organ and transplanted subcutaneously again into the mice. Only mice that received the tung metastases developed solid tumors and pulmonary metastases. Thereafter, these pulmonary metastases were serially transplanted subcutaneously into Balb/c mice. The solid tumors of the 7th passage were excised under sterile conditions and the cells were then cultured in serum-free medium (3) followed by colonization in soft agar. One of the clones derived from a colony was designated as r/mHM-SFME1. The r/mHM-SFME-1 cells were no longer resistant to G418. The profiles of the two cell lines in culture are shown in Figure 1. The r/mHM-SFME-1 cells tended to aggregate in culture whereas the ras/myc SFME cells did not. Aggregates always appeared 3 4 days after plating whether in serum-free or in serum-supplemented media and did not disappear upon adding fresh media. The aggregates sometimes detached from cells on dishes so that freely floating cells were observable in aged cultures even maintained by frequent media change. In order to confirm that the r/mI-LM-SFME-1 cells were derived from the ras/myc SFME cells, we determined whether the r/mHMSFME-1 cells had human ras genes. Hind III-digested fragments of DNA from r/mHM-SFME-1 and ras/myc SFME ceils were hybridized with the human c-Ha-ras exon-2 (6). A plasmid pUCC-H-ras, which contains normal human ras gene from placenta, for probe was obtained from the Japanese Cancer Research Resources Bank (JCRB). Six bands were detected in each of the fragments and no significant differences in the band profiles of either fragment were observed (Fig. 2). A faint band detectable in the DNA fragments from non-transformed SFME cells may be due to endogenous

Aberrant methylation of secreted apoptosis-related protein 2 (SARP2) in pure pancreatic juice in diagnosis of pancreatic neoplasms.

Objective: Secreted apoptosis-related protein (SARP) families are considered to counteract the oncogenic Wnt signaling pathway, and inactivation of this gene may aid cancer development and progression. Recently, the aberrant methylation of SARP2 was detected frequently in pancreatic carcinoma (PCa) tissue, but not in normal pancreatic tissue. We evaluated the hypermethylation of SARP2 in pure pancreatic juices (PPJ) aspirated endoscopically from patients with PCa, intraductal papillary mucinous neoplasm of the pancreas (IPMN), chronic pancreatitis (CP), and a control group who were consequently free of pancreatic diseases according to the differential diagnosis of PCa. Methods: We investigated the aberrant methylation of SARP2 in 9 human PCa cell lines and in the PPJ samples from 33 patients with PCa, 20 with IPMN, 19 with CP, and 10 control patients. DNAs extracted from not only sediment, but also the supernatant of the PPJ and PCa cell lines were treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction (PCR) (MSP). Moreover, real-time MSP was also performed for the melting curve analysis and the quantitative analysis of SARP2 in the PPJ. Results: The incidence of the aberrant methylation of SARP2 using MSP was 8/9 (89%) in PCa cell lines, 26/33 (79%) in the PPJ with PCa, and 17/20 (85%) with IPMN. However, it was only 1/19 (5%) in the PPJ with CP, and 0/10 (0%) in the PPJ of the control patients, respectively. The incidences of aberrant methylation of SARP2 in the PPJ with PCa and IPMN were significantly higher than that in the PPJ with CP (P < 0.001, P < 0.001). Melting curve analysis by real-time MSP revealed that the incidences of aberrant methylation of SARP2 in PPJ was 28/33 (85%) with PCa, 9/11 (82%) with the malignant group of IPMN, 5/9 (56%) with the benign group of IPMN and 5/19 (26%) with CP. In this analysis, there were significant differences between PCa and CP (P < 0.001), and between the malignant group of IPMN and CP (P < 0.005). In the quantitative analysis by real-time MSP with a suitable cut-off value, the incidences of aberrant methylation of SARP2 in the PPJ with PCa, the malignant group of IPMN, the benign group of IPMN, and CP were 19/33 (58%), 6/11 (55%), 3/9 (33%), and 2/19 (11%), respectively. The incidence of the aberrant methylation of SARP2 in the PPJ was significantly different between PCa and CP and between the malignant group of IPMN and CP (P < 0.005 and P < 0.05, respectively). Conclusions: Hypermethylation of SARP2 in the PPJ may be a highly sensitive and useful marker for the detection of pancreatic neoplasms, including PCa and the malignant group of IPMN.

Expression of vacuole membrane protein 1 (VMP1) in spontaneous chronic pancreatitis in the WBN/Kob rat.

Objectives: VMP1 is a stress-induced gene that is overexpressed in acute pancreatitis. Its overexpression promotes the formation of intracellular vacuoles and cell death. We investigated the expression of VMP1 mRNA and its relation to apoptosis in spontaneous chronic pancreatitis in the WBN/Kob rat. Methods: Four-week-old male WBN/Kob rats were fed a special breeding diet, MB-3, for 20 weeks. Rats were killed every 4 weeks, and the pancreas was examined. VMP1 mRNA expression was determined by reverse transcriptase polymerase chain reaction with a semiquantitative analysis, direct sequencing, and in situ hybridization. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) were used to detect cell proliferation and apoptosis, respectively. Results: Vacuolar formation was most prominent at 12 weeks, when chronic pancreatitis occurred. VMP1 mRNA was also strongly expressed at 12 weeks. In situ hybridization revealed VMP1 mRNA was expressed in acinar cells. Apoptosis was increased at 12 and 20 weeks, and PCNA expression was strongest at 16 weeks in the course of chronic pancreatitis. Conclusions: VMP1 mRNA expression paralleled the formation of vacuoles and apoptosis in acinar cells in the course of chronic pancreatitis in WBN/Kob rats.

Multiple factors influencing the release of hTERT mRNA from pancreatic cancer cell lines in in vitro culture

Since telomerase expression is highly prevalent in human cancers, the quantitation of serum/plasma hTERT (human telomerase reverse transcriptase) mRNA levels may be useful for early detection of PCa (pancreatic cancer). To analyse the correspondence between exhTERT (extracellular hTERT) mRNA levels and hTERT expression, we designed a cell culture system to investigate factors modulating the extracellular levels of hTERT mRNA in media conditioned by eight PCa cell lines. We found that the level of exhTERT mRNA was dependent on cell growth rate. MIAPaCa‐2, PANC‐1, KLM‐1 and PK‐9 cells expressed high levels of exhTERT mRNA, independent of cell density, whereas proliferating PK‐59, BxPC‐3 and PK‐45H cells released low levels of exhTERT mRNA. The augmented release of mRNA by spontaneous dead MIAPaCa‐2 cells was further increased at postconfluence. In Capan‐1 cells, low correspondence of marker was also due to RNase secretion. Upon reaching confluence, some PCa cell lines showed down‐regulation of hTERT expression. Following cell—cell adhesion, as shown by E‐cadherin engagement, PK‐59 cells showed levels of extracellular message below the limits of detection, a loss not due to an increase in message degradation. These results suggest that the levels of exhTERT mRNA in the medium of PCa cell lines are altered not only in response to cell growth rate and cell destruction, but are responsive to extracellular cues such as RNases and cell density. A cell‐free assay for exhTERT mRNA may therefore not be useful for early detection of PCa.

Application of the polymerase chain reaction (PCR) to quantify micro-metastasis in an experimental animal

In vitro cultured r/mHM-SFME-1 cells were injected into the hind foot pads of Balb/c mice. Metastasis was detected in the lungs of tumor-bearing mice by means of both PCR and histological methods. Primers for the PCR were set to amplify a 128 bp exon-1 sequence of the human c-Ha-ras1 gene which had been introduced into the cells. Resulted PCR bands were densitometorically quantified using a bioimage analyzer, and more than 1 x 10(4) tumor cells were detectable in the mouse lung. The number of tumor cells per lung estimated from the amount of PCR products was 1 x 10(5), 15 x 10(5), 1 x 10(5) and 40 x 10(5) on days 7, 14, 21 and 28 respectively after the tumor injection. No metastases were histologically observed on days 7 and 14. Then, the possibility of using this model system for evaluation of a treatment against micro-metastases is discussed.

Non-transformed, but not ras/myc-transformed, Serum-free Mouse Embryo Cells Recover from Growth Suppression by Azatyrosine

The anti‐proliferative effect of azatyrosine, a newly discovered antibiotic from Streptomyces, was examined in Balb/c‐originated serum‐free mouse embryo (SFME) cells and transformed ras/myc SFME cells which have activated human c‐Ha‐ras genes. Azatyrosine suppressed their growth in a concentration‐dependent manner. Growth suppression in both cells was detectable within 2 days after culture with 250 μg/ml azatyrosine. Non‐transformed SFME cells, however, regained rapid growth after 6 days even in the presence of azatyrosine, whereas ras/myc SFME cells did not recover from the suppression. Despite the growth inhibition of ras/myc SFME cells, expression of human ras in the cells was not inhibited by azatyrosine. Meanwhile, SFME cells have the ability to express glial fibrillary acidic protein (GFAP). This expression is induced by serum‐supplemented medium, though the serum inhibits the growth of SFME cells. Azatyrosine did not induce GFAP in ras/myc SFME cells, but inhibited growth. Furthermore, azatyrosine did not induce GFAP in SFME cells, and had no effect upon the expression of GFAP induced by serum in these cells. These results suggest that azatyrosine inhibited the growth of ras/myc SFME cells through a mechanism independent of those involved in growth inhibition and induction of GFAP expression by serum in SFME cells.

Detection of Micro-Metastasis by Polymerase Chain Reaction (PCR).

In order to establish validation system of micro-metastasis in r/mHM-SFME-1 cells, syngeneic Balb/c mice subcutaneously received the cells. DNA samples were prepared from the lungs of tumor-bearing mice and a PCR was employed to detect metastasized cells. Primers for the PCR were set to amplify a 128 bp exon-1 sequence of human c-Ha-rasl which had been stably transfected in the cells. Amplified bands were densitometorically quantified, so that 1x104 or more tumor cells were detectable in a mouse lung. The numbers of metastasized tumor cells per lung estimated by densitometer on day 7, 14, 21 and 28 of injection were 1x105, 7x105, 1x105 and 45x105 respectively, whereas no metastases were observed in histological sections on days 7 and 14.