The role of telomerase for pancreatic carcinogenesis in human and hamster

Telomerase activity and terminal restriction flagment (TRF) length were investigated in human and hamster pancreatic duct adenocarcinomas. In the hamster primary and transplantable pancreatic carcinomas and cell lines, telomerase activity increased 86 to 215.7 times relative to the levels in normal spleen and pancreas, and reduction of TRF length was observed. In 38 human pancreatic ductal carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymph node involvement and distant metastasis. These results suggest that telomerase play an important role for pancreatic duct carcinogenesis.     

Recall of obstetric events by mothers of schizophrenic patients

Telomerase activity and terminal restriction fragment (TRF) length were examined in hepatocellular carcinoma (HCC). Telomerase activity was assayed by telomeric repeat amplification protocol (TRAP) connected with an internal telomerase assay standard (ITAS). The incidence of strong telomerase activity (highly variable level compared with the activity of non-cancerous liver tissue) was 79% in well, 84% in moderately, and 100% in poorly differentiated HCC, while 0% in non-cancerous liver tissues. The incidence of TRF length alteration (reduction or elongation) was 53% in HCC. The incidence of TRF alteration was significantly higher in HCC exceeding 3 cm in diameter, moderately or poorly differentiated in histology. Telomerase activity was not associated with TRF length alteration in HCC. In conclusion, strong telomerase activity and TRF length alteration increased with HCC tumor progressions.     

Semi-quantitative analysis of telomerase in pancreatic ductal adenocarcinoma

Using a polymerase chain reaction-based amplification assay, we measured telomerase activity in surgically resected pancreatic ductal carcinomas (n = 16 cases) and normal ducts (n = 6), comparing findings with the telomerase activity of a human pancreatic cancer cell line, MIA PaCa-2, as a standard, i.e., relative telomerase activity was determined. Telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of MIA PaCa-2 per microgram protein of tissue samples. The median value for telomerase activity in normal pancreatic ducts was 0.13 and the 25th and 75th percentile were 0.01 and 0.76. The median value for telomerase activity in pancreatic ductal adenocarcinoma was 34.7 (25th percentile, 4.98; and 75th percentile, 296), significantly higher than that of normal ducts (P < 0.001). When the cut-off value was set at 1.0 and 3.0, the telomerase positivity rate of pancreatic ductal adenocarcinomas was 100% and 81.3%, respectively. Telomerase may be specific marker for pancreatic ductal carcinomas.     

Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung

Mutations of the K-ras gene have been implicated in the pathogenesis of human lung adenocarcinomas. In most studies published so far, squamous cell lung cancers harbored ras mutations only exceptionally or no mutations were detected at all. We have examined 141 lung tumor DNA samples for mutations in codons 12, 13, and 61 of K-ras and H-ras oncogenes. A large panel of 118 squamous cell carcinomas was included in the study. For K-ras codon 12, we used a sensitive two-step PCR-restriction fragment length polymorphism method which detects <1% of mutated DNA in the sample. K-ras mutations were found in 17 tumors (12%; 14 in codon 12 and 3 in codon 13). Among 19 adenocarcinomas, mutation was revealed in 7 samples (37%). Of these, one sample harbored two point mutations in codon 12. Nine mutational events were found in squamous cell carcinomas (8%, one adenosquamous carcinoma included, all in codon 12). Of four large cell carcinomas, one contained a mutation. Mutant-enriched PCR products harboring mutations were directly sequenced. Fifteen mutational events were G-->T transversions or G-->A transitions, one was a G-->C transition, and one sample revealed a frameshift deletion of one G from codon 12. Similar mutational spectrum was found in both squamous cell carcinomas and adenocarcinomas, suggesting similar carcinogenic pathways in both histological types of the tumor. The presence of mutations did not correlate with the stage of the disease. Moreover, we analyzed all samples for mutations in codons 12, 13, and 61 of the H-ras gene. We found only one mutation in codon 12. Thus, H-ras mutations apparently play an inferior role in lung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not only lung adenocarcinomas but also of a subset (about 8%) of squamous cell carcinomas.     

Decrease of telomere length in thyroid adenomas without telomerase activity

In somatic cells, telomeres shorten with population doubling, thus limiting their capacity to divide. Telomerase, which synthesizes telomeric repeats, can compensate for such shortening. Telomerase activity is known to be absent from most somatic differentiated cells but is present in germline cells, immortal cell lines, or a large majority of malignant tumors. Autonomous thyroid adenomas are benign tumors composed of highly differentiated cells characterized by TSH-independent function and growth. Telomere length and telomerase activity were measured in autonomous and hypofunctioning adenomas and their surrounding tissues. A significant decrease of 3.8+/-1.0 kilobases (kb) was observed in the length of the terminal restriction fragments (TRF) in 12 autonomous adenomas (8.6+/-1.1 kb), compared with the TRF length of their surrounding tissues (12.4+/-1.6 kb). The same kind of decrease, 3.5+/-1.2 kb, was also observed in 16 hypofunctioning adenomas (12.3+/-1.7 kb in surrounding tissue and 8.8+/-1.6 kb in the adenomas). No telomerase activity was detected either in the 12 autonomous adenomas studied or in most of the quiescent tissues (10 of 12). Most of the hypofunctioning adenomas tested (15 of 16) did not display telomerase activity. These results suggest that the cells have undergone a higher number of cell divisions in the adenomas than in the surrounding tissue. Moreover, there is a larger spread of the TRF length distribution in autonomous adenomas than in the collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule, some of which have reached the end of their life span, whereas others are still proliferating (but with no malignant potential for the autonomous adenomas). In conclusion, benign adenomas exhibit a shorter and more variable telomere length than the normal collateral quiescent tissue, with no telomerase activity to compensate this loss in telomere length.     

Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver? A molecular analysis of 101 liver lesions from 93 patients

It can be difficult to distinguish benign bile duct proliferations (BDPs) from well-differentiated metastatic peripancreatic adenocarcinomas on histological grounds alone. Most peripancreatic carcinomas harbor activating point mutations in codon 12 of the K-ras oncogene, suggesting that K-ras mutational status may provide a molecular basis for distinguishing BDPs from liver metastases. The ability of tests for mutations in codon 12 of K-ras to make this distinction was examined in a two-part study. In the first part we determined the K-ras mutational status of 56 liver lesions and 48 primary peripancreatic adenocarcinomas obtained from 48 patients. In the second part of this study an additional 45 liver lesions were studied. In the first 48 patients, activating point mutations in codon 12 of K-ras were detected in 28 (61%) of the 46 primary carcinomas, in 8 (100%) of 8 liver metastases, in 2 (6.5%) of 31 BDPs, and in none (0%) of 14 liver granulomas. Three BDPs and two primary carcinomas did not amplify. To further estimate the prevalence of K-ras mutations in BDPs we analyzed an additional series of 45 mostly incidental BDPs for K-ras mutations. Three (6.7%) of these 45 harbored K-ras mutations. These results suggest that K-ras mutations may be useful in distinguishing BDPs from metastases in the liver; however, there is some overlap in the mutational spectra of BDPs and pancreatic carcinomas.     

Comparison of oncogene mutation detection and telomerase activity for the molecular staging of non-small cell lung cancer

Novel oncogene mutation detection techniques have demonstrated that standard histopathological examination may fail to detect clinically significant metastatic cancer cells. Recently, telomerase activity has been detected in most immortal cell lines and human tumors, potentially providing a novel diagnostic marker. We compared standard histopathological examination with the telomeric repeat amplification protocol assay and either a p53 plaque hybridization or a K-ras mutation ligation assay in the lymph nodes of 12 patents with surgically resectable non-small cell lung cancer. Telomerase activity was detected in 10 of 10 (100%) evaluable tumors. Eight of 9 (89%) histopathologically positive lymph nodes were telomerase positive, and 26 of 48 (54%) histopathologically negative lymph nodes were telomerase positive. In comparison, oligonucleotide plaque hybridization detected metastases in all 3 histopathologically positive nodes and in 3 of 27 histopathologically negative nodes. Similarly, the K-ras mutation ligation assay detected metastases in all 6 histopathologically positive lymph nodes examined and in 1 of 21 histopathologically negative lymph nodes. Thus, most of the "positive" nodes by telomerase assay did not harbor occult neoplastic cells that shared the same genetic alteration as the primary tumor. The high rate of false positives associated with the telomeric repeat amplification protocol assay limits its role in staging lymph nodes in patients with non-small cell lung cancer.     

Telomerase activity in colorectal carcinoma and its relationship to clinicopathological findings

Telomerase is thought to be responsible for cell immortality. The telomerase activity in carcinomas has been remarked since 1995. We examined telomerase activity in colorectal carcinoma by TRAP (Telomeric repeat amplification protocol) assay, and investigated its relationship to clinicopathological findings. We could analyse telomerase activities in 33 cases (66%) of 50 colorectal carcinomas, whereas the activity was not found in all 13 cases of noncancerous colorectal mucosa. There was no relation between the telomerase activity and the clinicopathological findings or metastatic status. We confirmed telomerase activities in much of colorectal carcinomas in spite of their progression. The carcinoma cells might be immortal from their early stage of progression by means of telomerase activity.     

In situ mRNA hybridization technique for analysis of human telomerase RNA in gastric precancerous and cancerous lesions

Telomerase, the ribonucleoprotein enzyme that elongates telomerase, is repressed in normal somatic cells but is reactivated during tumor progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human gastric precancerous and cancerous lesions by using in situ mRNA hybridization (ISH) with avidin-biotin staining. We also examined telomerase activity in these lesions by using hybridization protection assay connected with a telomeric repeat amplification protocol (TRAP/HPA). Analyzed tissue samples were as follows; 132 cases of chronic atrophic gastritis without intestinal metaplasia, 115 incomplete-type intestinal metaplasias, 40 complete-type intestinal metaplasias, 23 hyperplastic polyps, 23 tubular adenomas and 26 adenocarcinomas. In ISH analysis, high levels of hTR expression were observed preferentially in the nuclei at the single-cell level. hTR-expressing cells in carcinomas and adenomas were significantly more frequent than those of the other lesions (P < 0.001). The expression pattern of hTR in carcinoma and adenoma tissues was heterogeneous and similar intratumor heterogeneity was detected in Ki-67 immunoreactivity. Infiltrating lymphocytes in tissue also exhibited high levels of hTR expression. In TRAP/HPA analysis, carcinomas had significantly more frequent positivity for telomerase activity and a higher level of telomerase activity than the other lesions (P < 0.05). However, the amount of telomerase activity did not parallel the expression level of hTR. Our data suggest that hTR expression increases in the early stages of stomach carcinogenesis and that sufficient synthesis of hTR is a prerequisite for telomerase reactivation in tumorigenesis.     

Synergistic effects of prostaglandin F2alpha and tumor necrosis factor to induce luteolysis in the pig

Telomerase activity was detected in germ cells, stem cells and cancer cells. In tumors of the ovary, an organ that contains germ cells, the authors examined availability to detect telomerase activity. Telomerase activity of malignant tumors was extremely high compared with that of normal ovaries and benign tumors. Strength and frequency of telomerase activity in malignant tumors was significant different from that in benign tumors. Telomere length tended to be smaller for malignant tumors of advanced stage, but no significant relationship between telomere length and telomerase activity and tumor stage could be recognized. Telomerase activity may be a useful marker for the diagnosis of ovarian tumors.     

The presence of K-12 ras mutations in duodenal adenocarcinomas and the absence of ras mutations in other small bowel adenocarcinomas and carcinoid tumors

BACKGROUND: Adenocarcinomas and carcinoid tumors are the most common malignant tumors of the small intestine. K-ras oncogene mutations at codon 12 are common in gastric, pancreatic, and colon carcinomas, with an incidence of 35-88%. K-ras mutations have not been extensively studied in either adenocarcinomas or carcinoid tumors of the small bowel. The purpose of this study was to determine whether ras mutations play an important role in the formation of these tumors. METHODS: Archival tissues from 28 adenocarcinomas and 22 carcinoid tumors of the small bowel were studied, along with archival tissues from 32 adenocarcinomas of the large bowel, which were used as controls. DNA from the small intestine tumors was analyzed for K-ras, H-ras, and N-ras oncogene mutations at codons 12, 13, and 61, using polymerase chain reaction and sequence specific oligonucleotide hybridization techniques. Large bowel adenocarcinomas were analyzed for K-ras mutations at codons 12 and 13. RESULTS: A point mutation of K-ras at codon 12 was detected in 4 of 28 (14.3%) of the small bowel adenocarcinomas, in 12 of 32 (37.5%) of the large bowel adenocarcinomas, and in 0 of 22 small intestine carcinoid tumors. No other K-ras, H-ras, or N-ras mutations were detected in any of the small bowel tumors. Each small intestine K-ras mutation was found in a duodenal adenocarcinoma (4 of 12 cases, 33%), whereas none occurred in 16 other jejunal or ileal adenocarcinomas. CONCLUSIONS: K-ras mutations appear to play a significant role in the pathogenesis of duodenal adenocarcinomas, but they do not appear to be important in the development of jejunal or ileal adenocarcinomas or of carcinoid tumors of the small intestine.     

Clinical significance of telomerase activity in precancerous lesion of the liver (adenomatous hyperplasia)

To understand the role of telomere dynamics in hepatocellular carcinogenesis, we examined the lengths of terminal restriction fragments (TRFs) in hepatocellular carcinoma (HCC) and surrounding tissues with chronic active hepatitis (CAH), liver cirrhosis (LC) and atypical adenomatous hyperplasia (AAH). The peak TRFs in all HCCs were significantly shorter than those of the surrounding tissues (CAH, LC). TRF in AAH was shortened and similar to that of HCC. Telomerase was examined in CAH, LC, AH, and HCC, and detected in high levels almost exclusively in HCCs. Interestingly, the intensity of telomerase activity in the AH was similar to that of HCC. Thus, the progressive shortening of telomere and the activation of telomerase may be a useful marker for the early detection of malignant progression in liver disease.     

Relationship between telomere length and clinical and biological characteristics of the cancers with telomerase reactivation

Activation of telomerase and stabilization of telomeres are considered to be necessary for immortalization of human tumor cells. Telomerase activity and telomere lengths were examined in adult and childhood cancer tissues. High telomerase activity was detected in over 40% samples. In these cases, the lengths of telomeres varied in wide range and the short telomere length significantly correlated with high proliferative index. The patients with short telomeres demonstrated poorer prognosis than other patients. These findings suggest that the short telomeres might be related with the malignant potential in cancers with high telomerase activity.     

Pyloric gland adenoma of the main pancreatic duct

A case of a pyloric gland type adenoma of the main pancreatic duct in a 69-year-old woman is reported. The tumor led to occlusion and cystic dilatation of the main duct in the pancreatic tail. The surgical resection specimen disclosed a polypoid, bilobed mass attached to the wall of the main pancreatic duct by a thin fibrous stalk. Light-microscopic examination revealed a well-demarcated nodule composed of closely packed tubular glands lined by columnar, mucin-secreting cells with abundant clear cytoplasm and basally oriented nuclei. Focal, mild cytologic atypia was seen. Pyloric metaplasia and focal papillary hyperplasia was present in the adjacent ductal epithelium. Periodic acid-Schiff reactions, with and without diastase predigestion, showed reactivity in the tubular glands, whereas alcian blue (pH 2.5) was negative. Immunohistochemical stains for chromogranin, serotonin, somatostatin, and gastrin failed to detect the respective antigens. Genetic analysis using polymerase chain reaction with mutant enrichment and allele specific oligonucleotide hybridization detected a single mutation at codon 12 of K-ras, which changed the wild-type glycine to arginine. This mutation is commonly found in invasive pancreatic ductal carcinomas. Although tumors with microscopic and immunohistochemical features consistent with pyloric gland adenoma have been described in the gallbladder, to our knowledge, this is the first reported case within the pancreatic ductal system. The finding of a K-ras, codon 12 mutation and the presence of focal dysplasia may denote neoplastic potential in association with this lesion.     

Telomerase and telomere length in the development and progression of premalignant lesions to colorectal cancer

Telomerase and telomere length are increasingly studied as prognostic markers in malignancy. Telomerase is also known to be expressed in certain nonmalignant cells, although generally at low levels. We investigated telomerase activity and telomere length in premalignant, malignant, inflammatory, and normal colon specimens to determine whether significant differences exist and whether telomerase may serve as a marker for early- or late-stage colorectal cancer. Telomerase activity was evaluated in 130 frozen specimens from human colon cancer (n = 50), adjacent normal colon tissue (n = 50), colon polyps (n = 20), and colitis (n = 10) using a modified telomeric repeat amplification protocol assay, and telomere length was assessed by terminal restriction fragment analysis. High to moderate levels of telomerase activity were detected in 90% of colorectal tumors. Weakly positive activity was detected in 10%. None of the normal tissues exhibited telomerase activity. In polyps and colitis, telomerase activity was found in 60% (12 of 20) and 40% (4 of 10), respectively. Telomerase activity in both nonmalignant lesions was 25- to 54-fold lower than that detected in colon cancer (P < 0.001). We found a positive correlation between tumor cell infiltration determined in cryostat sections and telomerase activity (r = 0.886; P > 0.0001). Late-stage tumors (Dukes C + D) demonstrated increased telomerase activity compared to early-stage tumors (Dukes A + B). Telomere restriction fragments in colon tumors had peak values of 4.8 +/- 1 kbp that were significantly and consistently shorter than those of the adjacent normal tissues (7.54 +/- 1.3 kbp), polyps (7.5 +/- 0.7 kbp), and colitis specimens (7.7 +/- 0.5kbp; P < 0.0001). Telomeres were 0.6 kbp longer in tumors with high telomerase activity and in late-stage cancers (Dukes C + D) compared to those in tumors with low telomerase activity and in early-stage cancers (Dukes A + B). Our data demonstrate that telomerase in colon cancer was commonly acquired, and activity was higher than that in polyps and colitis. However, weak telomerase activity was detected in premalignant and inflammatory lesions. Telomeres in colon cancer were considerably shorter, an indication of extensive cell proliferation and population divisions, whereas adjacent normal colon specimens, polyps, and colitis had comparable telomere lengths. Our results indicate that increased telomerase activity occurs in colon cancer cells that have undergone extensive telomere shortening relative to surrounding normal tissues and in which telomerase-induced stabilization of telomeres may be critical for the continued proliferation of the malignant clone. The link between telomerase activity and stage suggests that telomerase is up-regulated as a function of increased tumor cell invasion, tumor progression, and metastatic potential in colon cancer.     

Telomerase activity in human laryngeal squamous cell carcinomas

The activation of telomerase has been shown to be an important step during tumorigenesis in a variety of malignancies and is associated with characteristics of cellular immortality, such as indefinite proliferative potential. We studied telomerase activity in a series of human laryngeal carcinomas. Thirty-six tumors from 35 patients were studied using a sensitive PCR-based technique, the telomeric repeat amplification protocol assay. Telomerase activity was present in 32 tumors (89%), and the level of activity correlated with the stage of disease. In two of four telomerase-negative tumors, we found evidence of an inhibitor of telomerase activity. In many cases, samples of mucosa surrounding the tumor were also studied, and telomerase could be detected in 16 of 21 patients. For this reason, we proceeded to perform a topographical analysis that demonstrated a pattern of telomerase activity suggestive of a spread of telomerase-positive cells. In conclusion, these data indicate that telomerase activation is important for laryngeal carcinogenesis and that telomerase assay might be a valuable addition to determine the spread of the disease.     

Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients

Mutated K-ras oncogenes have been detected in a third of lung adenocarcinomas, located usually in codon 12, its presence correlating negatively with survival. To further define the role of K-ras point mutations in non-small cell lung cancer, we studied the presence of mutated K-ras genes in surgical specimens from 66 patients. Polymerase chain reaction was performed from sections of formalin-fixed paraffin-embedded tissue. We screened for point mutations in codons 12, 13 and 61 of the K-ras gene by dot blot hybridization analysis with mutation-specific oligonucleotide probes. Ras gene mutations were present in 13 of 66 carcinomas (20%), nine in codon 12 and four in codon 61. Three squamous cell carcinomas harbored two different point mutations in K-ras codon 12. Mutated K-ras genes were found more frequently in squamous cell carcinomas (eight of 38) than in adenocarcinoma (three of 22). Analysis of nucleotide sequence disclosed a multifarious mutation pattern of K-ras codon 12, where the most common conversion was from glycine (GGT) to valine (GTT). K-ras point mutation positive subset had poorer survival, nine of the 13 patients died during the follow-up period as compared with 22 of 53 patients with no mutation in the K-ras gene (P = 0.01). The difference was also strikingly significant when stratified according to node status.     

Detection of K-ras gene mutations at codon 12 in the pancreatic juice of patients with intraductal papillary mucinous tumors of the pancreas

BACKGROUND: The authors previously found specific mutations of the K-ras gene at codon 12 in the pancreatic juice of 67% of patients (6 of 9) with pancreatic ductal carcinoma, and the detection of these mutations was useful for diagnosis. This study was performed to detect and evaluate K-ras mutations in pancreatic juice from patients with intraductal papillary mucinous tumor of the pancreas, which is considered a low grade malignancy. The results were interpreted from the viewpoint of clinical significance. METHODS: K-ras mutations were examined using seminested polymerase chain reaction analysis combined with restriction enzyme digestion, followed by nonradioisotopic single strand DNA conformation polymorphism. RESULTS: Twelve of thirteen cases (92%) of intraductal papillary mucinous tumor of the pancreas, confirmed histologically (9 adenomas and 4 carcinomas), and 26 of 43 cases (60%) of ductal carcinoma showed specific K-ras gene mutations in the pancreatic juice. Furthermore, 4 of 22 patients (18%) with chronic pancreatitis, followed for more than 1 year without a sign of pancreatic tumor, showed K-ras mutations. In contrast, no mutations of the K-ras gene were detected in the pancreatic juice from 28 normal controls. CONCLUSIONS: K-ras mutations were found in the pancreatic juice of all but one patient with intraductal papillary mucinous tumor of the pancreas, but they were not useful for distinguishing carcinoma from adenoma. The authors concluded that K-ras mutations are not a specific marker for pancreatic neoplasms because similar mutations were detected in the pancreatic juice from patients with chronic pancreatitis. At the present time, the detection of K-ras mutations in pancreatic juice should be used clinically as an adjunct diagnostic modality for pancreatic diseases.     

Telomerase activity and telomere lengths in various cell lines: changes of telomerase activity can be another method for chemosensitivity evaluation

For the cancer cells which have overcome the second mitotic clock (M2), activated telomerase is essential and used as another marker of immortality. Many trials had been initiated to target telomerase, which is known to be specific to tumors. To determine the best in vitro cell system for testing the efficacy of telomerase inhibitors, we evaluated the telomerase activity of various cancer cell lines and measured their telomere lengths. We also treated some cancer cell lines with adriamycin and measured the changes of telomerase activity. Telomerase activity was evaluated in various cell lines with the TRAP (telomeric repeat amplification protocol) assay. Telomerase activity was calculated and translated into arbitrary units by computer-assisted densitometry with the control of telomerase activity in the 293 control cell line. Also, terminal restriction fragment lengths were measured using Southern blotting. We also measured telomerase activity and telomere lengths in 11 benign breast tumor tissues and 19 paired stomach cancer and normal tissues. Cancer cell lines treated with adriamycin we evaluated for changes of telomerase activity and the cell proliferation by MTT assay and dye exclusion test. Telomerase activity of cell lines was 95.3 24.1 unit with a range of 27.6-129.6 unit, while the telomere lengths of those cell lines were variable from 5.0 to 10.4 kbp with a median of 6 kbp. In 11 cancer cell lines which were not yet firmly established, we could not detect any telomerase activity. Low telomerase activity was detected in only 2 benign tumor tissues of breast with a median telomere length of 8.8 (7-10.5) kbp. Among paired 19 gastric cancer and normal tissues, only 7 cancer tissues showed weak telomerase activity. After adriamycin treatment, telomerase activity in YCC-S-1, YCC-S-3, MCF-7 and MCF-7/ADR was decreased in accordance with the changes of the cell numbers. Telomerase is specific to cancer tissues and is expressed differently from organ to organ. Telomerase activity by TRAP assay could be used as a chemosensitivity assay.