Alternative RNA splicing in expression of the glutathione synthetase gene in human cells.

Ubiquitous free radical production occurs continuously in cells and tissues. Glutathione is the most abundant mammalian antioxidant, and is synthesized by glutathione synthetase (GSS). Therefore, GSS plays an important role in defending the cell against reactive oxygen species. The expression of GSS has been studied in human cells; however, sequence information about alternative splicing variants of GSS mRNA has not been reported. In the present study, we identified a novel alternative splicing variant (ASV) of the GSS gene in 10 human normal tissues and five human cancer cell lines. The deleted transcript of GSS was characterized by an in-frame deletion of 333 bp, corresponding to the complete loss of exons 4 and 5. Thus this GSS ASV causes protein truncation. We quantified the mRNA of GSS ASV in human normal tissues using real-time PCR. The ASV was detected in colon, kidney, lung, liver, placenta, peripheral blood and uterus, but not in heart, skeletal muscle and spleen tissue. Our results provide a basis for more detailed studies on the regulation of GSS, and for further evaluation of this and other possible roles of GSS. Understanding the regulation of GSS expression is very important for the development of new strategies for controlling the development of GSH-based redox homeostasis.

Laparoscopic choledochotomy in management of choledocholithiasis.

PURPOSE: Laparoscopic choledochotomy on patients indicated for common bile duct exploration was carried out according to an algorithm for managing choledocholithiasis. This study describes retrospectively our method and evaluates a new cystic duct biliary decompression cannula (J-tube) as an alternative to the T-tube. METHODS: Patients with confirmed choledocholithiasis (n=46) underwent laparoscopic choledochotomy. The T-tube was inserted in cases with suspected retained stones after common bile duct clearance, and the J-tube (950-mm long, 4 Fr) with a tapered and J-shaped segment at the distal end was inserted in other cases. RESULTS: Only 1 case was converted to open surgery (success rate, 97.8%); the J-tube was inserted in 30 patients and the T-tube in 15. The median operation time, hospital stay, and the interval until removal of the tube were significantly shorter with J-tube than with T-tube cases. Bile leakage after surgery occurred in 4 J-tube and 2 T-tube cases with one residual stone in each case. CONCLUSIONS: The transcystic decompression tube is easily and safely inserted with the J-kit. Among several strategies currently available for the management of choledocholithiasis, laparoscopic choledochotomy with the use of the J-tube is one of the safest and most feasible methods.

[A case of long-term survival of gastric cancer with peritoneal metastasis effectively treated by TS-1 and paclitaxel (PTX) combination therapy].

We experienced the case of a 62-year-old woman who obtained long-term survival of three years by TS-1+paclitaxel (PTX) administration for gastric cancer postoperative peritoneal metastases. We first performed total gastrectomy, and the diagnosis was T3N2M0, Stage IIIB. Next, we performed chemotherapy by postoperative 5-FU+cisplatin (CDDP) and met in ambulatory. A bowel movement aberration was found during the course at 3 years postoperatively, and close inspection revealed sigmoid colon stenosis by peritoneal metastases. After construction of an artificial anus, we started administration of TS-1+paclitaxel. Chemotherapy was continued on an outpatient basis, and at-home treatment was possible for about 26 months till symptoms aggravated. No grave adverse event occurred except for depilation of grade 1. It was thought that this long-term treatment on an outpatient basis contributed to survival.

Expression of hTERT mRNA in a mortal liver cell line during S phase without detectable telomerase activity.

Normal human liver cells have a limited capacity for proliferation due to telomere shortening, whereas immortalized cells prevent shortening of the 3' single strand telomeric repeat by expressing telomerases, including human telomerase reverse transcriptase (hTERT). The hTERT transcript contains three deletion sites that give rise to alternatively spliced variants (ASVs). Recently, hTERT expression was observed in cycling primary presenescent human fibroblasts, which were believed to lack hTERT expression and telomerase activity. hTERT mRNA was expressed in the synthesis (S) phase of the cell cycle. Although hTERT mRNA has eight isoforms, it is not known which of the hTERT ASVs are expressed in S phase. In order to determine the possible relationships between the cell cycle and ASV expressions, we measured the full-length isoform and ASVs of hTERT mRNA in a mortal liver cell line and immortal cell lines that were synchronized in S phase of the cell cycle. Using RT nested-PCR analysis, the full-length isoform and alpha-deletion ASV of hTERT were detected in the LI90 mortal liver cell line at points when cells in S phase represented >48% of the cell population without detectable telomerase activity. hTERT was always expressed in the HLE and Huh-7 hepatocellular carcinoma cell lines, regardless of the cell cycle. Our results suggest the possibility that telomerase is regulated in a cell cycle-dependent manner in normal liver cells.

Expression profile of a gamma-deletion variant of the human telomerase reverse transcriptase gene.

The human telomerase reverse transcriptase (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of chromosomes. The hTERT transcript has been shown to have two deletion type alternative splicing sites. One deletion site induces the alpha-deletion variant, lacking 36 bp from exon 6, and the other induces the beta-deletion variant, lacking 182 bp from exons 7 and 8. Here, we identified a novel deletion variant of the hTERT transcript in hepatocellular carcinoma cell lines. The deleted transcript was characterized by an in-frame deletion of 189 bp, spanning nucleotides 2710 to 2898, corresponding to the complete loss of exon 11 (gamma-deletion). The region lacking in the gamma-deletion lies within RT motifs D and E, suggesting that it is missing conserved residues from the catalytic core of the protein. Both gamma- and alpha-deletion variants were occasionally detected, but the beta-deletion variant was frequently observed. Our results may provide important information for more detailed studies on the regulation of telomerase activity.

Genetic detection for hematogenous micrometastasis in patients with various types of malignant tumors using Uroplakin II derived primers in polymerase chain reaction.

The presence of circulatory metastasis is one of the most significant factors for poor-prognosis in patients with several types of cancer. To establish a sensitive reverse transcription PCR assay to detect micrometastasis in blood containing several cancer types, we first investigated Uroplakin II (UP II), a novel molecular marker for human transitional cell carcinoma of the bladder, in 25 types of normal organs. In our study, UP II mRNA was detected in 10 types of organs, including bladder, kidney, lung and pancreas, but was not detected in normal lymph nodes or leukocytes. The data indicated evidence of UP II expression in various types of normal tissues by RT-nested PCR analysis. UP II mRNA was detected in 2 of 11 (18.2%) peripheral blood samples from lung cancer patients with no metastasis, and in 5 of 12 (41.7%) peripheral blood samples of lung cancer patients with metastasis. UP II was also detected in 6 of 16 (37.5%) peripheral blood samples of patients with pancreatic cancer. The data are particularly important in that the molecular detection of micrometastasis in the blood by means of UP II mRNA identification is feasible for UP II-positive neoplasms, including lung and pancreatic cancers.

Expression of molecular marker genes in various types of normal tissue: implication for detection of micrometastases.

Many researchers have investigated the expressions of candidates for a suitable reverse transcription nested polymerase chain reaction (RT-PCR) marker. But typically biomarkers often have false-positive results. We assessed whether epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSM) could be detected in 28 different types of normal human sources. Using RT-nested PCR assay, EGFR mRNA was also detected in various types of normal tissue, including pancreas, prostate and uterus. CEA was detected in various types of normal tissue, including prostate, uterus, bladder and spleen. PSM mRNA was also detected in various types of normal tissue, including kidney, liver, skeletal muscle, spleen, bladder and ovary. We report here that the expression of these biomarkers in normal cells might have induced false-positives, and that further enhancement of sensitivity might compromise specificity. Conversely, these biomarkers can be utilized for attempts to define micrometastases in various types of tumors whose cells express these tissue-specific genes.

Telomerase activity and hTERT mRNA in development and progression of adenoma to colorectal cancer.

Telomerase activity and hTERT mRNA expression are upregulated in colorectal cancer. Whether they are inherent in colorectal adenomas, premalignant lesions to cancer, however, remains to be elucidated. We examined telomerase activity by the fluorescence-based telomeric repeat amplification protocol method and analyzed the level of hTERT mRNA by real-time polymerase chain reaction in 74 surgically obtained neoplasms from 29 patients. The specimens were divided into 6 categories according to the criteria of the Vienna Classification. The control comprising 29 non-pathological mucosa were classified into category 1, 6 adenomas indefinite for neoplasia into category 2, 21 non-invasive low grade adenomas into category 3, 23 high grade adenomas or non-invasive carcinomas into category 4, and 15 intramucosal or submucosal carcinomas into category 5. Carcinoma invading beyond the submucosa (9 samples) was referentially subdivided into category 6. Telomerase activity (mean +/- standard error) in 1 categories to 6 were 5.0+/-1.2, 1.8+/-1.7, 4.3+/-1.6, 20.2+/-2.1, 36.4+/-5.5, and 55.5+/-8.2 units/microg protein, respectively. There were no statistical differences between categories 1 and 2, 1 and 3, and 2 and 3. A significant statistical difference in the other two was observed by the multiple comparison test. The mean levels of hTERT mRNA was 103.1+/-102.4, 103.6+/-103.0, 103.6+/-102.9, 103.7+/-102.9, 104.0+/-103.4, and 104.4+/-104.0 copies/microg total RNA, respectively. There was a significant statistical difference only between category 6 and each of the other categories. These results suggest that telomerase activation occurs during the progression from low-grade to high-grade dysplasia in adenomas and increases steadily with the progression of the degree of dysplasia and invasion during colorectal carcinogenesis, and that hTERT mRNA expression is a feature of the late stage development of colorectal cancer.

Adenosquamous cell carcinoma arising from the papilla major.

A 47-year-old man was admitted to hospital with complaint of general fatigue. Shortly before the admission a suspected obstructive jaundice was diagnosed at a local hospital. On admission, the physical examination was significant for jaundice; total bilirubin was 6.43 mg/dl. The tumor marker CA19-9 was 2056 U/ml. Endoscopic retrograde cholangiopancreatography (ERCP) was performed and showed dilatation of common bile duct and main pancreatic duct, accompanied with an endoscopic naso-biliary drainage (ENBD) in order to reduce the jaundice. The duodenoscopy showed enlarged and deformed papilla. Hypotonic duodenography showed a filling defect at the medial side of the second portion of the duodenum. Ultrasonography (US) showed a hyperechoic lesion, sized 15 mm in diameter, at the pancreas head with dilatation of biliary tract and main pancreatic duct. An abdominal enhanced CT scan showed a mass sized 15 mm at the lower edge of the common bile duct. A selective hepatic arteriography showed no special finding. We performed a pancreatoduodenectomy with dissection of the lymph nodes. The tumor, sized 22x15x20 mm, was white colored and solid on the papilla. Histopathological inspection of the specimen showed an adenosquamous cell carcinoma of the bile duct in the papilla. The tumor was found to infiltrate the neighboring pancreas and to contain metastasis in lymph nodes in the hepatoduodenal ligament, post pancreaticoduodenal and para-aortic lymph nodes. This is the first report on a case of adenosquamous carcinoma of the papilla major.