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.

Effect of gemcitabine on the expression of apoptosis-related genes in human pancreatic cancer cells.

AIM: To investigate the expression of genes involved in the gemcitabine-induced cytotoxicity in human pancreatic cancer cells. METHODS: A human pancreatic cancer cell line, PANC-1, was cultured. 1x10(4) PANC-1 cells were plated in 96-well microtiter plates. After being incubated for 24 h, gemcitabine was added to the medium at concentrations ranging 2.5-1000 mg/L. The AlamarBlue dye method was used for cell growth analysis. DNA fragmentation was quantitatively assayed using a DNA fragmentation enzyme-linked immunosorbent assay (ELISA) kit. PAP and TP53INP1 mRNA expression was determined using the reverse transcription-polymerase chain reaction with semi-quantitative analysis. The expression of GSK-3beta and phospho-GSK-3beta proteins was examined with Western blot analysis. RESULTS: The IC50 for the drug after a 48-h exposure to gemcitabine was 16 mg/L. The growth of PANC-1 cells was inhibited by gemcitabine in a concentration-dependent manner (P<0.0001) and the cell growth was also inhibited throughout the time course (P<0.0001). The DNA fragmentation rate in the gemcitabine-treated group at 48 h was 44.7%, whereas it was 25.3% in the untreated group. The PAP mRNA expression was decreased after being treated with gemcitabine, whereas the TP53INP1 mRNA was increased by the gemcitabine treatment. Western blot analysis showed that phospho- GSK-3beta (ser9) was induced by the gemcitabine treatment. CONCLUSION: Gemcitabine suppresses PANC-1 cell proliferation and induces apoptosis. Apoptosis is considered to be associated with the inhibition of PAP and GSK-3beta, and the activation of TP53INP1 and pospho-GSK-3beta (ser9).

Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer.

AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer. METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed. RESULTS: TP53INP1 was expressed in 98% (139/142 cases) of non-cancerous gastric tissues and was down-expressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%). Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3% vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients). P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P=0.0006). CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.

Oral vaccination of mice against rodent malaria with recombinant Lactococcus lactis expressing MSP-1(19).

AIM: To construct the recombinant Lactococcus lactis as oral delivery vaccination against malaria. METHODS: The C-terminal 19-ku fragments of MSP1 (MSP-1(19)) of Plasmodium yoelii 265-BY was expressed in L. lactis and the recombinant L. lactis was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P. yoelii 265-BY parasites of erythrocytic stage. The protective efficacy of recombinant L. lactis was evaluated. RESULTS: The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8+/-0.4% and 20.8+/-26.5%, respectively, and those of their control groups were 12.0+/-0.8% and 60.8+/-9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment. However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well. CONCLUSION: The results imply the potential of recombinant L. lactis as oral delivery vaccination against malaria.

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.

Tumor protein p53-induced nuclear protein 1 (TP53INP1) in spontaneous chronic pancreatitis in the WBN/Kob rat: drug effects on its expression in the pancreas.

CONTEXT: The tumor protein p53-induced nuclear protein 1 (TP53INP1) gene was found using DNA microarray technology as an overexpressed gene in acute pancreatitis. However, expression of TP53INP1 in chronic pancreatitis has not been previously reported. OBJECTIVE: This study investigated TP53INP1 gene expression and its relationship with p53 and apoptosis in spontaneous chronic pancreatitis in the Wistar-Bonn/Kobori rat. METHODS: Ninety four-week-old male Wistar-Bonn/Kobori rats were fed a special breeding diet until sacrifice. Camostat mesilate (n=30) or a herbal medicine (Saiko-keishi-to; n=30) were mixed with the diet, while the other 30 rats were untreated. The rats were sacrificed every 4 weeks for 20 weeks, and the pancreas was examined. In addition, 6 four-week-old male Wistar-Bonn/Kobori rats were sacrificed and studied as starting reference. Finally, Wistar rats (n=36) were studied as controls. MAIN OUTCOME MEASURE: TP53INP1 mRNA expression was determined by reverse transcription-polymerase chain reaction using semi-quantitative analysis, direct sequencing and in situ hybridization. RESULTS: TP53INP1 mRNA was strongly expressed at 12 weeks when chronic pancreatitis developed, with a second peak at 20 weeks. The expression kinetics of TP53INP1 mRNA paralleled acinar cell apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The p53 mRNA expression showed a single peak at 12 weeks. In situ hybridization revealed that TP53INP1 mRNA was expressed mainly in acinar cells. Therapeutic drugs such as camostat mesilate and a herbal medicine Saiko-keishi-to suppressed the TP53INP1 mRNA expression. TP53INP1 mRNA induction in acinar cells was confirmed with in vitro experiments using an arginine-induced rat pancreatic acinar AR4-2J cell injury model. CONCLUSIONS: TP53INP1 expression may reflect the acute-phase response and apoptosis of acinar cells in the course of chronic pancreatitis.

Regulation of aroP expression by tyrR gene in Escherichia coli.

tyrR gene encodes a global regulatory protein (TyrR), which plays an important role in the transcriptional regulation of eight transcription units (including tyrR gene itself) whose protein products catalyze key steps in aromatic amino acid biosynthesis and/or transport. The aroP gene encodes an integral membrane protein (AroP) that transports aromatic amino acids through the cell membrane. The transcription of aroP was reported to be repressed by TyrR. In this work, aroP(p) (aroP gene carrying its own promoter), aroP (aroP gene without promoter) and tyrR genes were amplified by PCR from genomic DNA of E. coli K12 and introduced into E. coli WT5. The expression of aroP and tyrR were detected and the activities of AroP and TyrR were determined. The introduction of either aroP(p) or aroP elevated the strain's transport activity by 1.40 or 1.46-fold respectively. Transformant carrying tyrR gene showed an ATPase activity 1.69-fold compared with the control. When the genes were linked in tandem and co-expressed in a plasmid, the relative AroP transport activity of the strain harboring aroP(p) -tyrR (0.95) was significantly lower than that of aroP-tyrR (1.31). The results indicated that TyrR might be able to reduce the expression of aroP gene by binding with the aroP promoter region in E.coli.

Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum.

AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production.

Effect of F209S Mutation of Escherichia coli AroG on Resistance to Phenylalanine Feedback Inhibition.

In Escherichia coli, 80% of the 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase was encoded by aroG gene. The aroG gene was amplified by polymerase chain reaction(PCR) from strain K-12 and a mutant strain resistant to phenylalanine analogues. The PCR products were cloned and subject to DNA sequence analysis. A single base mutation of Tright curved arrow C was detected at nucleotide 625, which causes a substitution of Phe(209) by Ser in the gene product. The gene was expressed on pTrc99A in E.coli strain JM105. Under the induction of IPTG, distinct band with the expected molecule weight was detected on SDS-polyacrylamide gel electrophoresis and the specific activity of DAHP of the crude extract of the transformed cells increased by 1.8-fold. Enzyme activity inhibition analysis revealed the high resistance of mutant AroG to feedback inhibition by phenylalanine. JM105 cells harboring with mutant aroG gene showed were able to grow on medium containing higher concentration of analogues than that carrying normal aroG gene. Discussion was focused on the varieties of mutations contributing to desensitization of feedback inhibition.

Cloning and Expression of aroG Gene of E. coli and Its Co-expression with pheA and tyrB Genes.

3-Deoxy-D-arabino-heptulonate-7-phosphate synthetase (DAHP) is one of the key enzymes in phenylalanine biosynthesis pathway. In E. coli, DAHP is encoded by aroG Gene. In this work, aroG was cloned from an E. coli mutant strain resistant to m-fluro-L-phenylalanine (mPF) and p-fluro-L-phenylalanine (pPF) by PCR. The gene was expressed under the control of lambda phage promoter p(R) in P2392 strain of E. coli. Distinct band was detected as the product of aroG on SDS-PAGE. The specific activity in crude extract of DAHP was raised to 1.7-fold. Based on the cloning and expression of pheA (encoding both chorsmate mutase CM and prephenate dehydratase PD) and tyrB (encoding phenylalanine aminotransferase PAT) genes, aroG, pheA and tyrB genes were constructed and expressed in P2392. The results showed that the specific activities of DAPH, CM/PD and PAT in crude extracts were increased by 1.7, 13.9/7.8 and 2.3-fold, respectively.