Hideaki Niiyama

Adenovirus-mediated p53 Gene Transduction Inhibits Telomerase Activity Independent of Its Effects on Cell Cycle Arrest and Apoptosis in Human Pancreatic Cancer Cells

Evidence for a relationship between overexpression of wild-type p53 and telomerase activity remains controversial. We investigated whether p53 gene transduction could cause telomerase inhibition in pancreatic cancer cell lines, focusing on the relation of transduction to growth arrest, cell cycle arrest, and apoptotic cell death. The cells were infected with recombinant adenovirus expressing wild-type p53 or p21WAF1 at a multiplicity of infection of 100 or were continuously exposed to 10 microM VP-16, which is well known to induce apoptosis. Adenovirus-mediated p53 gene transduction caused G1 cell cycle arrest, apoptosis, and resultant growth inhibition in MIA PaCa-2 cells; the cell number 2 days after infection was 50% of preinfection value, and 13% of the cells were dead. Moreover, the transduction resulted in complete depression of telomerase activity through down-regulation of hTERT mRNA expression. In contrast, p21WAF1 gene transduction only arrested cell growth and cell cycle at G1 phase, and VP-16 treatment inhibited cell growth with G2-M arrest and apoptosis; after treatment, the cell number was 73% of pretreatment, and 12% of the cells were dead. Neither p21WAF1 gene transduction nor VP-16 treatment caused telomerase inhibition. Similar results were obtained in two other pancreatic cancer cell lines, SUIT-2 and AsPC-1. Thus, our results demonstrate that the p53 gene transduction directly inhibits telomerase activity, independent of its effects on cell growth arrest, cell cycle arrest, and apoptosis.

Quantitative analysis of hTERT mRNA expression in colorectal cancer

OBJECTIVES:Telomerase is highly activated in a variety of malignant neoplasms including colon cancer. Among the major components of telomerase, human telomerase reverse transcriptase (hTERT) is thought to regulate telomerase activity. To assess the importance of telomerase for the diagnosis of colorectal cancer, we measured the expression of hTERT mRNA and telomerase activity in a large series of 140 colorectal cancers, 140 adjacent normal tissues, and 20 adenomas.METHODS:The expression level of hTERT was measured quantitatively by competitive reverse transcriptase–polymerase chain reaction (RT-PCR), and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in the same samples.RESULTS:The median expression level of hTERT mRNA in carcinomas was significantly higher than that in either adenomas or normal tissues. The median level of hTERT in adenomas was significantly higher than that in normal tissues. Telomerase activities in carcinomas were significantly higher than those in either adenomas or normal tissues. Telomerase activities in adenomas were also significantly higher than those in normal tissues. Furthermore, the relative expression levels of hTERT mRNA in adenomas and carcinomas were significantly correlated with the relative telomerase activities; the Spearman rank correlation was 0.53 (p = 0.021) and 0.18 (p = 0.031), respectively.CONCLUSIONS:Our data suggest that determination of hTERT mRNA by competitive RT-PCR is superior in quantitative accuracy and sensitivity and would support the importance of telomerase activity for the diagnosis of colorectal cancer.

Telomerase activity detected in pancreatic juice 19 months before a tumor is detected in a patient with pancreatic cancer

We report a patient with pancreatic cancer in whom telomerase activity had been detected in the pancreatic juice 19 months before he was diagnosed as having pancreatic cancer. A 61-yr-old alcoholic man complaining of epigastric and back pain was diagnosed as having groove pancreatitis based on the presence of inflammation in the pancreatic head and its extension to the duodenal mucosa with an associated elevated serum amylase level. All imaging modalities showed no sign of a tumor. However, high telomerase activity was detected in the pancreatic juice collected during endoscopic retrograde pancreatography. His symptoms subsided due to abstinence from alcohol. A tumor, however, was recognized on computed tomography 19 months later, at which time the patient immediately underwent a pylorus-preserving pancreaticoduodenectomy. The carcinoma was located mainly in the Santorini duct region. High telomerase activity in the pancreatic juice may precede clinical detection of pancreatic cancer and thus could be a useful early diagnostic marker for pancreatic cancer.

9-Hydroxyellipticine inhibits telomerase activity in human pancreatic cancer cells

There is increasing interest in identifying potent inhibitors of telomerase because the enzyme plays a crucial role in the development of cellular immortality and carcinogenesis. We hypothesized that 9‐hydroxyellipticine (9‐HE), an antitumor alkaloid, would inhibit telomerase activity because the drug has a unique mechanism of inhibiting phosphorylation of mutant p53 protein via inhibition of protein kinases, thereby restoring wild‐type p53 function. This study was conducted to examine the effect of 9‐HE on telomerase activity in human pancreatic cancer cells with differing p53 gene status. 9‐HE treatment at relatively high concentrations resulted in rapid, complete inhibition of telomerase activity, irrespective of the p53 status. We conclude that 9‐HE may exert a strong inhibitory effect on telomerase activity possibly through inhibition of protein kinases rather than through restoration of functional wild‐type p53.

Composite glandular-endocrine cell carcinoma of the extrahepatic bile duct: Immunohistochemical study

Summary A composite glandular‐endocrine cell carcinoma of the extrahepatic bile duct in a 64 yr old Japanese man is reported. A nodular polyp measuring 1.9×1.1 cm was located in the confluence of the extra‐hepatic bile duct. Histologically, the tumor was composed of well differentiated tubular adenocarcinoma and small cell neuroendocrine carcinoma with a transition between the 2 components. The 2 areas of the tumor immunohistochemically revealed a clear‐cut difference in functional differentiation. Tumor cells in the glandular component were immunoreactive to both carcinoembryonic antigen (CEA) and CAM 5.2, while those in the small cell area were immunoreactive to neuroendocrine markers such as neuron specific enolase (NSE), chromogranin A and serotonin. These results suggest that the tumor arose from a multipotential stem cell capable of differentiation in 2 directions.

Possible role of telomerase activation in the multistep tumor progression of periampullary lesions in patients with familial adenomatous polyposis

OBJECTIVES:The role of telomerase in periampullary tumor progression in patients with familial adenomatous polyposis (FAP) was investigated.METHODS:Relative telomerase activity was measured using a telomerease amplification protocol in periampullary biopsy specimens of normal mucosa and adenoma obtained from patients with FAP, and was compared with that of periampullary normal mucosa and cancer specimens from patients without FAP.RESULTS:None of normal mucosa from the non-FAP patients showed a telomerase ladder. Telomerase was positively detected in three of seven normal mucosa (42.9%) and in five of seven adenoma from FAP patients (62.5%). In papillary cancer from the non-FAP patients, seven of nine tissue specimens (77.8%) showed positive activity. When semiquantitatively analyzed, the relative telomerase activity increased in accordance with the progression of the diseases.CONCLUSIONS:Telomerase is activated even in normal mucosa of FAP patients, and the intensities of telomerase may reflect the malignant potential of periampullary neoplasms.

Diverse effects of 9-hydroxyellipticine on the chemosensitivity of human pancreatic cancer cells harboring p53 mutations

Recently, it has been shown that 9-hydroxyellipticine (9-HE), an antitumor alkaloid has a unique property of restoring functional wild-type (wt) p53 activity via inhibition of mutant (mt) p53 protein phosphorylation. In the present study, we investigated the effect of 9-HE on the drug sensitivity of human pancreatic cancer cells. Exposure of cells to 9-HE at a relatively low concentration of 1 microM induced almost no cell death but was sufficient to restore wt p53 activity, as evidenced by an induction of endogenous p21WAF1/CIP1 concomitant with G1 and G2/M arrests in cell-cycle progression. Pretreatment with 1 microM 9-HE markedly enhanced cell killing when combined with cisplatin or mitomycin C. In contrast, 9-HE pretreatment protected cells from killing by 5-fluorouracil, VP-16, or vincristine. These effects of 9-HE were specific for several cell lines containing mt p53 and were not observed in p53-negative or wt p53 expressing cells. Taken together, these findings suggest that 9-HE may exert different effects on the drug sensitivity of pancreatic cancer cells displaying p53 mutations possibly through restoration of wt p53.