Differential gene expression of human telomerase-associated protein hTERT and TEP1 in human hematopoietic cells

The maintenance of telomere length is crucial for the survival of cells. Recently, genes for proteins that consist of human telomerase have been cloned and the results have indicated a close relationship between telomerase activity and its gene expression. We studied the mRNA expression of the telomerase-associated genes, hTERT and TEP1, in hematopoietic cells in order to clarify the relation between them and telomerase activity using semiquantitative RT-PCR. In polymorphonuclear cells and monocytes isolated from peripheral blood, which had no detectable telomerase activity, no hTERT mRNA expression was seen. On the other hand, lymphocytes and CD34-positive cells both demonstrated hTERT mRNA expression. TEP1 mRNA was detected in all samples, showing no differential expression. We then assessed hTERT and TEP1 mRNA expression in CD34-positive cells cultured in vitro with growth factors. After 4 weeks of culture, all the cells showed myeloid differentiation and the telomerase activity was downregulated. hTERT mRNA was expressed in CD34-positive cells, but was downregulated in 4-week-cultured cells. TEP1 showed no apparent differential expression. We conclude that hTERT mRNA expression is downregulated in accordance with telomerase downregulation during the course of myeloid differentiation, which suggests that it plays a crucial role in the expression of enzyme activity, while TEP1 has a much smaller role to play, if any.     

Detection of mutations of p53 tumor suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer: comparison with K-ras mutation.

Because of the difficulty in obtaining biopsy specimens from pancreatic cancer patients, K-ras mutation analysis in pancreatic juice has been used for specific diagnosis. But recently, false positives have been obtained with this method. To improve the genetic diagnosis of pancreatic cancer, detection of p53 gene mutation in pancreatic juice was studied. Pancreatic juice was sampled endoscopically. Single-strand conformation polymorphism analysis was used for p53 mutation analysis. Furthermore, K-ras mutations at codon 12 were also studied in the same pancreatic cancer patients. Of 26 cases of pancreatic cancer, p53 mutations were detected in 11 (42.3%). No mutations were seen in the cases with mucin-producing adenoma nor with chronic pancreatitis. K-ras mutations were detected in 84.0% of cases by RFLP analysis, which has high sensitivity, and in 65.3% by hybridization protection assay, which has high specificity. Using a combination assay with both genes, genetic abnormalities were detected in 92.0% by RFLP and 73.1% by hybridization protection assay including two cases in which p53 alone was positive by both methods. The specificity of p53 mutation for pancreatic cancer is very high. Therefore, simultaneous analysis of p53 and K-ras mutation is suggested to enhance the genetic diagnosis of pancreatic cancer.     

K-ras point mutations in the supernatants of pancreatic juice and bile are reliable for diagnosis of pancreas and biliary tract carcinomas complementary to cytologic examination.

In order to clarify whether DNA analysis for K-ras mutation can be used to diagnose cancers in supernatants of pancreatic juice and bile, samples from 29 cases of pancreatic, biliary tract, gastric, and neuroendocrine carcinomas, 1 malignant lymphoma case, 2 cases of pancreatic adenoma, 9 cases of chronic pancreatitis and 21 other non-cancer cases were examined. Polymerase chain reaction (PCR) products for K-ras gene codons 2 to 97 of exons 1 and 2 were generated with 33/33 (100%) pancreatic juice and 41/41 (100%) bile samples. By the single strand conformation polymorphism (SSCP) method, point mutations were detected in the pancreatic juice or bile supernatants of 13/13 (100%) pancreas cancer cases, 5/14 (35.7%) biliary tract cancer cases, 1/2 (50.0%) pancreatic adenoma cases and 3/9 (33.3%) chronic pancreatitis cases. Direct sequencing confirmed identical point mutations in the supernatants, malignant cells of cytologic smears of pancreatic juice or bile and cancer tissues. The DNA analysis demonstrated the presence of K-ras point mutations in 3 cases of pancreatic carcinomas with false-negative cytologic diagnoses. This novel method allows simultaneous testing for genetic abnormalities in supernatants of pancreatic juice and bile, after removing cells for cytologic diagnosis and screening for pancreatic and biliary tract tumors.     

Clinical significance of k-ras and c-erbB-2 mutations in pancreatic adenocarcinoma and chronic pancreatitis

Background: The differentiation of chronic pancreatitis (CP) from pancreatic adenocarcinoma (PA) remains the great challenge for clinicians. The purpose of this study was to compare the prevalence of K-ras and c-erbB-2 mutations in PA and CP in order to evaluate their usefulness in differential diagnosis of those diseases. Methods: The study included 49 patients who underwent Whipple resection or distal pancreatectomy for pancreatic adenocarcinoma (26 subjects) or chronic pancreatitis (23 subjects). DNA from pancreatic tissue was analyzed for K-ras codon 12 and c-erbB-2 mutations with PCR amplifications. Results: The K-ras gene mutation has been shown in 20 (76.9%) PA cases and in 8 (34.8%) CP cases (p<0.01). Prevalence of c-erbB-2 amplification in patients with PA was 17 (65.3%), which was not different from CP, 16 (56.5%) (p=0.58). There was a significant correlation between K-ras mutation and lymph node metastases (p=0.025) as well as between K-ras mutation and G3 tumor differentiation (p=0.037). Overall median survival in patients with PA was 9.5 mo. There was no relationship between presence of K-ras (p=0.58) or c-erbB-2 (p=0.17) mutation and survival time in PA patients. Conclusion: Those results may indicate that both K-ras and c-erbB-2 play a role in pancreatic carcinogenesis, however only K-ras may provide an additional tool in differential diagnosis of CP and PC.     

Activation of the hTERT expression in squamous cell cervical carcinoma is not associated with gene amplification

The hTERT gene encodes the telomerase catalytic subunit that plays a key role in cancer cell immortalization. Earlier, hTERT amplification was detected in squamous cell cervical carcinomas (SCC), however possible relations between elevated hTERT mRNA level and gene amplification was not studied. Here, we compared the hTERT expression and copy number in the same tumors by quantitative real-time PCR. The hTERT DNA copy number was virtually unchanged in all 33 studied tumors, when compared to normal tissues. This result was confirmed using two reference genes beta-actin and beta-D-glucuronidase. Nevertheless, the activation of hTERT expression was found in 80% of cases (37/46, p<0.001). There was no correlation between the degree of mRNA increase and the tumor size and/or presence of metastases. No hTERT gene expression was observed in 20% of cases (9/46), while the control GADPH expression was unchanged. The detected elevation of the hTERT mRNA level was found using primers specific to functionally active full-length isoform of mRNA. Similar results were obtained with SCC cell lines carrying human papilloma virus (HPV) genomes. We conclude that frequent activation of hTERT expression in SCC is not associated with gene amplification.     

Comparative analysis of K-ras point mutation,telomerase activity,and p53 overexpression in pancreatic tumours

K-ras point mutation, p53 over-expression, and telomerase activity have been proposed as molecular markers for clinical diagnosis of pancreatic carcinoma. To evaluate the clinical usefulness of these markers, we performed comparative analysis in 61 resected pancreatic samples including 15 intraductal papillary-mucinous tumours (IPMTs), 4 mucinous cystic tumours, 37 ductal adenocarcinomas, and five chronic pancreatitis samples. K-ras point mutation, telomerase activity, and p53 overexpression were analyzed using mutant allele specific amplification, the telomeric repeat amplification protocol, and immunohistochemical staining, respectively. In malignant tumours, K-ras mutation, telomerase activity, and p53 overexpression were detectable in 76, 91, and 46%, respectively, while in benign tumours, these alterations were detectable in 38, 0, and 0%, respectively. Among 15 IPMTs, K-ras mutation was detectable in 4 (80%) of 5 IPMT-adenomas, 4 (80%) of 5 IPMT-carcinomas and 2 (66%) of 3 papillary-mucinous carcinomas, which are invasive carcinomas derived from IPMTs. Telomerase activity was not detectable in IPMT-adenomas, but was detected in all 5 IPMT-carcinomas and 3 papillary-mucinous carcinomas. p53 overexpression was not detected in IPMTs, but was detected in 2 (66%) of 3 papillary-mucinous carcinomas, indicating that telomerase is likely to be activated concomitant with carcinogenesis. These results suggest that telomerase activity is the most useful as a differential diagnostic marker between malignant and benign pancreatic tumours.     

食管鳞癌组织端粒酶亚基的表达及端粒酶活性的关系

目的　探讨食管鳞癌组织中端粒酶活性、端粒酶逆转录酶 (h TERT)及端粒酶相关蛋白 - 1(TP- 1)的表达及其关系。方法　应用 TRAP-银染法对 45例食管鳞癌组织端粒酶活性的检测 ,同时应用原位杂交对癌组织切片进行 h TERT、TP- 1的 m RNA表达的检测。结果　癌组织端粒酶活性阳性率为 82 .2 %。癌组织中不同分化程度的端粒酶活性差异无显著性 (P>0 .0 5 )。有淋巴结转移者癌组织端粒酶活性明显高于无淋巴结转移者 ,差异有显著性(P<0 .0 5 )。癌组织中 h TERTm RNA表达的阳性率为 6 4.4%,TP- 1的阳性率为 6 2 .2 %。 h TERT的 m RNA表达与端粒酶活性密切相关 ,而 TP- 1的 m RNA表达与端粒酶活性无相关。结论　食管鳞癌组织中端粒酶活性及h TERT、TP- 1的 m RNA表达均较高。端粒酶活性与淋巴结转移有关。 h TERT与端粒酶活性有密切关系。     

k-ras Point Mutations in the Supernatants of Pancreatic Juice and Bile Are Reliable for Diagnosis of Pancreas and Biliary Tract Carcinomas Complementary to Cytologic Examination

In order to clarify whether DNA analysis for K-ras mutation can be used to diagnose cancers in supernatants of pancreatic juice and bile, samples from 29 cases of pancreatic, biliary tract, gastric, and neuroendocrine carcinomas, 1 malignant lymphoma case, 2 cases of pancreatic adenoma, 9 cases of chronic pancreatitis and 21 other non-cancer cases were examined. Polymerase chain reaction (PCR) products for K-ras gene codons 2 to 97 of exons 1 and 2 were generated with 33/33 (100%) pancreatic juice and 41/41 (100%) bile samples. By the single strand conformation polymorphism (SSCP) method, point mutations were detected in the pancreatic juice or bile supernatants of 13/13 (100%) pancreas cancer cases, 5/14 (35.7%) biliary tract cancer cases, 1/2 (50.0%) pancreatic adenoma cases and 3/9 (33.3%) chronic pancreatitis cases. Direct sequencing confirmed identical point mutations in the supernatants, malignant cells of cytologic smears of pancreatic juice or bile and cancer tissues. The DNA analysis demonstrated the presence of K-ras point mutations in 3 cases of pancreatic carcinomas with false-negative cytologic diagnoses. This novel method allows simultaneous testing for genetic abnormalities in supernatants of pancreatic juice and bile, after removing cells for cytologic diagnosis and screening for pancreatic and biliary tract tumors.     

Preoperative diagnosis of intraductal papillary-mucinous tumors of the pancreas with attention to telomerase activity

BACKGROUND: It has been reported that, in patients with intraductal papillary-mucinous tumor (IPMT) of the pancreas, it is difficult to distinguish adenoma from carcinoma preoperatively. Recently, it has also been reported that telomerase activity was detected in many patients with carcinoma. In this report, the authors used the method of telomerase repeat amplification protocol (TRAP) assay on pancreatic juice retrieved by endoscopic retrograde pancreatic juice aspiration (ERP aspiration). METHODS: Pancreatic juice was collected from 28 patients (13 with intraductal carcinoma and 15 with adenoma) using ERP aspiration at either Hiroshima University Hospital or its affiliated hospitals. Two samples of pancreatic juice were collected from each patient. Each sample was examined by cytology for Papanicolaou staining and TRAP assay. RESULTS: Four of 13 IPMT patients (31%) with intraductal carcinoma were diagnosed accurately by cytology. Seven of nine patients who were classified with benign tumors by cytologic assessment had tumors that expressed telomerase activity. Overall, 11 of 13 IPMT patients (85%) with intraductal carcinoma were diagnosed correctly by cytology associated with telomerase activity. All of the IPMT patients with adenoma were classified with benign tumors by cytologic assessment, and telomerase activity was not expressed. CONCLUSIONS: In this study, the authors found that telomerase activity was expressed with a comparatively high probability in intraductal carcinoma. These results suggest that telomerase activity in pancreatic juices may be used as an adjunct to cytologic diagnosis and may aid further in distinguishing between benign IPMT and malignant IPMT of the pancreas preoperatively.     

Dysregulated expression of the major telomerase components in leukaemic stem cells.

Telomere loss is rapid during the progression of chronic myeloid leukaemia (CML) and correlates with prognosis. We therefore sought to measure expression of the major telomerase components (hTR and hTERT) in CD34+ cells from CML patients and normal controls, to determine if their altered expression may contribute to telomere attrition in vivo. High-purity (median 94.1%) BCR-ABL+ CD34+ cells from CML (n=16) and non-CML (n=14) patients were used. CML samples had a small increase in telomerase activity (TA) compared to normal samples (approximately 1.5-fold, P=0.004), which was inversely correlated with the percentage of G0 cells (P=0.02) suggesting TA may not be elevated on a cell-to-cell basis in CML. Consistent with this, hTERT mRNA expression was not significantly elevated; however, altered mRNA splicing appeared to play a significant role in determining overall full length, functional hTERT levels. Interestingly, Q-RT-PCR for hTR demonstrated a mean five-fold reduction in levels in the chronic phase (CP) CML samples (P=0.002), raising the possibility that telomere homeostasis is disrupted in CML. In summary, the molecular events regulating telomerase gene expression and telomere maintenance during the CP of CML may influence the disease progression observed in these patients.     

Molecular target-based therapy of pancreatic cancer

Pancreatic cancer is genetically complex, and without effective therapy. Mutations in the Kirsten-ras (K-ras) oncogene occur early and frequently (approximately 90%) during pancreatic cancer development and progression. In this context, K-ras represents a potential molecular target for the therapy of this highly aggressive cancer. We now show that a bipartite adenovirus expressing a novel cancer-specific apoptosis-inducing cytokine gene, mda-7/interleukin-24 (IL-24), and a K-ras AS gene, but not either gene alone, promotes growth suppression, induction of apoptosis, and suppression of tumor development mediated by K-ras mutant pancreatic cancer cells. Equally, the combination of an adenovirus expressing mda-7/IL-24 and pharmacologic and genetic agents simultaneously blocking K-ras or downstream extracellular regulated kinase 1/2 signaling also promotes similar inhibitory effects on the growth and survival of K-ras mutant pancreatic carcinoma cells. This activity correlates with the reversal of a translational block in mda-7/IL-24 mRNA in pancreatic cancer cells that limits message association with polysomes, thereby impeding translation into protein. Our study provides support for a "dual molecular targeted therapy" involving oncogene inhibition and selective cancer apoptosis-inducing gene expression with potential for effectively treating an invariably fatal cancer.