Telomerase reverse transcriptase and telomeric-repeat binding factor protein 1 as regulators of telomerase activity in pancreatic cancer cells.

Telomerase adds hexameric repeats of 5'-TTAGGG-3' termed telomeres to ends of chromosomal DNA. This enzyme has been implicated in cellular immortalization and cellular senescence. Recently, a number of relevant genes have been cloned, including these encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein-1 (TEP1). Also important are genes encoding human telomeric-repeat binding factor protein (TRF) 1 and 2. To clarify mechanisms regulating telomerase activity, we studied telomerase activity, the telomeric restriction fragment (TRF) length and gene expression of these telomerase components and the telomeric-repeat binding factor proteins in sequential observation following X-irradiation of cultured pancreatic cancer cells. We previously reported that PANC-1 cells are better able to tolerate thermal stress, antineoplastic drugs, and exposure to tumour necrosis factor than MIAPaCa-2 cells. MIAPaCa-2 and PANC-1 cells were exposed to X-irradiation, their telomerase activity was increased at 2 days and then decreased gradually. Of the three telomerase components, only hTERT mRNA expression showed parallel changes. TRF length was stable just before and after X-irradiation. Among binding factor proteins, TRF1 mRNA showed reciprocal changes possibly directed toward maintaining a stable telomere length. In this study, our results demonstrate that not only hTERT but also TRF1 are important regulator of telomerase activity.

Expression of telomerase-associated genes: reflection of telomerase activity in gastric cancer?

Telomerase activation is a characteristic of immortalized tumor cells but not of normal cells. Telomerase activity has been detected in approximately 85% of malignant tumors, and assaying for telomerase activity is thought to be useful for diagnosing cancer. Three telomerase-associated molecules [human telomerase RNA component (hTR), telomerase-associated protein (TEP1), and human telomerase reverse transcriptase (hTERT)] have been cloned. We semiquantitatively measured telomerase activity and the expression of these genes in cancerous and noncancerous regions of gastric cancer patients. We also investigated whether the expression of these genes correlated with telomerase activity. Telomerase activity in cancerous regions was significantly higher than in noncancerous regions, but there was no correlation between telomerase activity and the expression of these genes. Furthermore, no clear difference was observed between cancerous and noncancerous regions. These data indicate that the level of three telomerase-associated genes (i.e., hTR, TEP1 mRNA, hTERT mRNA), do not reflect telomerase activity, and the RNA levels of these genes are not useful markers for diagnosing gastric cancer.

Role of human telomerase reverse transcriptase and telomeric-repeat binding factor proteins 1 and 2 in human hematopoietic cells.

Telomerase, an enzyme that adds hexameric repeats of 5'-TTAGGG-3', termed telomeres, to the ends of chromosomal DNA, has been implicated in cellular immortalization and cellular senescence. Recently several relevant genes have been cloned, including those encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein-1 (TEP1). Also important are genes encoding human telomeric-repeat binding factor proteins (TRF) 1 and 2. We compared 10 human malignant hematopoietic cell lines, 19 samples from patients with acute leukemia and normal granulocytes and monocytes to study telomerase activity and expression of these various genes using a reverse transcription-polymerase chain reaction (RT-PCR). In all 10 malignant cell lines with telomerase activity, hTR, hTERT mRNA, and TEP1 mRNA were expressed, while in normal monocytes and granulocytes without telomerase activity, expression of hTR, but not hTERT mRNA was detected. TEP1 mRNA was expressed in normal monocytes, but not granulocytes. Expression of TRF1 and TRF2 mRNAs was greater in the normal cells than in human malignant hematopoietic cell lines and in 16 samples of patients with acute leukemia. When differentiation of the malignant hematopoietic cell line HL-60 was induced using tumor-necrosis-factor 471 and all-trans retinoic acid (ATRA), telomerase activity decreased gradually during differentiation. Of the three telomerase components, only hTERT mRNA expression showed changes paralleling telomerase activity, becoming undetectable with differentiation. In contrast, initially low expression of TRF1 and TRF2 mRNAs increased during differentiation. Not only hTERT, but also TRF1 and TRF2 are important regulators of telomerase activity that represent potential targets for gene therapy against cancer.

Helicobacter pylori infection: augmentation of telomerase activity in cancer and noncancerous tissues.

Telomerase adds hexameric repeats of 5'-TTAGGG-3' to the ends of chromosomal DNA (telomere) and has been implicated in cell immortalization and cellular senescence. The aim of this study was to measure quantitatively the telomerase activity and human telomerase RNA component (hTR) content in gastric cancer and to examine the relation between these values and histologic factors including Helicobacter pylori as a risk factor for gastric cancer. Telomerase activity was measured by a modified telomeric repeat amplification protocol in cancerous and noncancerous tissues (intestinal metaplasia, chronic gastritis, normal mucosa) from 27 gastric cancer patients; hTR expression was examined by the quantitative reverse transcriptase-polymerase chain reaction using fluorescent probes. Telomerase activity was higher in cancers (total product generated: 33.7) than in noncancerous tissues. Telomerase activity was higher in intestinal metaplasia (16.7) and chronic gastritis (10.6) than in normal mucosa (3.5). In patients with intestinal-type gastric cancer, telomerase activity was higher in intestinal metaplasia with H. pylori infection than in that without infection. hTR expression was not correlated with telomerase activity. H. pylori infection may influence telomerase activity in cancer and noncancerous tissues.

Neoadjuvant intra-arterial infusion chemotherapy combined with hormonal therapy for locally advanced breast cancer.

To determine the efficacy of combined neoadjuvant intra-arterial infusion chemotherapy and hormonal therapy for treating locally advanced breast cancer, we compared the outcomes of patients with or without this therapy, and also assessed histologic response. Ninety-four patients with locally advanced breast cancer (stage IIIa, 56; stage IIIb, 38). Nineteen stage IIIa and 17 stage IIIb patients received intra-arterial plus hormonal therapy while 37 stage IIIa and 21 stage IIIb patients with similar ages and follow-up durations did not. Treated patients received intra-arterial epirubicin plus oral medroxy-progesterone. Five-year disease-free survival rates were 77.5% for intra-arterially treated and 33.0% for other patients in stage IIIa, and 70.5% for intra-arterially treated and 38.1% for other patients in stage IIIb. Five-year overall survival rates were 94.4% for intra-arterially treated and 61.7% for other patients in stage IIIa, and 90.9% for intra-arterially treated and 56.3% for other patients in stage IIIb. Ten-year overall survival rates in stage IIIb were 90.9% for treated and 22.5% for other group patents. All differences were statistically significant (p<0.05). Good histologic response to intra-arterial therapy was seen in 75% of the primary tumors and 71% of involved lymph nodes. Neoadjuvant intra-arterial therapy with hormonal therapy yielded better survival rates than no intra-arterial therapy or our previous intra-arterial regimen.

Telomerase activity is down regulated via decreases in hTERT mRNA but not TEP1 mRNA or hTERC during the differentiation of leukemic cells.

BACKGROUND: Recent studies have determined several telomerase-associated molecules, but the precise mechanisms regulating telomerase activity by those molecules has not been fully understood. MATERIALS AND METHODS: The telomerase activity was determined by TRAP assay. Using TaqMan RT-PCR, the quantitative and kinetic values of mRNA expression of the three telomerase-associated molecules were examined in HL60 cells differentiated with tumor necrosis factor mutant and all-transretinoic acid. RESULTS: The levels of telomerase activity in leukemic cell lines, leukemic cells from patients, and normal peripheral blood cells were distributed over a very wide range. Human telomerase catalytic subunit (hTERT) mRNA expression declined to nearly undetectable levels more rapidly than the inhibition of telomerase activity after treatment with these reagents in HL60 cells. Telomerase-associated protein (TEP1) mRNA increased approximately 6-fold over its level in untreated cells. The levels of human telomerase RNA component (hTERC) also increased approximately 2.7-fold at 5 days after treatment. CONCLUSIONS: These results suggest that telomerase activity is down-regulated mainly via decreases in hTERT, but not TEP1 and hTERC expression during the differentiation of leukemic cells.

Limitations of urinary telomerase activity measurement in urothelial cancer.

The reported frequency of detectable telomerase activity in spontaneously voided urine samples from patients with urothelial cancer varied from 0 to 85%. We examined stasis in the bladder and specimen storage as interfering conditions in this assay. Telomerase activity in exfoliated cells was measured by a polymerase-chain-reaction-based assay in spontaneously voided urine from urothelial cancer patients. Effects of retention in the bladder and specimen storage from voiding to measurement of telomerase activity were modeled by suspending 10(6) cells from the cancer-derived T24 line in normal urine (pH 6.5) at 37 degrees C and 25 degrees C, respectively. Hematuria was modeled by adding hemoglobin. In T24 cells suspended in urine at 37 degrees C, telomerase activity had decreased to approximately 20% of preincubation activity after 1 h, and had disappeared after 3 h. In urine at 25 degrees C, telomerase activity in T24 cells had decreased to approximately 40% of preincubation activity at 1 h and to <10% at 6 h. When we examined telomerase activity in exfoliated cells in spontaneously voided urine from urothelial cancer patients (excluding first-voided morning specimens), telomerase activity was detected in only 21% of samples (four of 19) despite measurement with 1 h of voiding and steps to avoid hemoglobin interference. Measurement of telomerase activity in spontaneously voided urine is insufficiently sensitive and reliable for the diagnosis of urothelial cancer.

Establishment of quantitative reverse transcription--polymerase chain reaction assays for human telomerase-associated genes.

Telomerase is an enzyme that synthesizes and adds repetitive telomeric sequences of (TTAGGG)n to the ends of chromosomes. Recently, several telomerase-associated genes have been cloned, making it possible to study the expression of these genes. Quantitative comparisons of the expression of these genes and of telomerase activity might help clarify the regulation of telomerase activity. Therefore, we established the validity of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the human telomerase catalytic subunit (hTERT) mRNA and telomerase associated protein (TEP1) mRNA using the TaqMan fluorogenic detection system. Using this assay, we quantitated hTERT mRNA and TEP1 mRNA expression in two human pancreatic cancer cell lines, AsPC-1 and PANC-1. Our results indicated that the levels of hTERT mRNA and TEP1 mRNA expression in AsPC-1 were 1.50 and 2.31 times higher than in PANC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the quantities of hTERT and TEP1 mRNAs in clinical specimens. Taken together, our results indicate that it is possible to measure the expression of the major telomerase genes subunits. Furthermore it is possible to apply this technique to determine the amount of other types of mRNA.

Neoadjuvant intraarterial high-dose chemotherapy and autologous peripheral blood stem cell transplantation for advanced breast cancer.

Eight locally advanced breast cancer patients were treated with neoadjuvant intraarterial high-dose chemotherapy (epirubicin) plus MPA combined with autologous PBSCT. All patients completed the scheduled treatment, and there were no toxic deaths. Patients were treated with an escalating dose of epirubicin (370-480 mg) and cyclophosphamide (0-6000 mg). The rate of clinical response was 100%. The rate of good histologic response was 87.5% in the main tumor and 75% in diseased lymph nodes. The 2-year survival rate was 100%. Six patients were disease-free at the time of writing. This treatment resulted in higher rates of clinical and histologic response when compared with standard-dose intraarterial chemotherapy.

Detection of human serum tumor necrosis factor-alpha in healthy donors, using a highly sensitive immuno-PCR assay.

BACKGROUND: Tumor necrosis factor-alpha (TNFalpha) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFalpha, including those in healthy controls, cannot be measured by existing methods. METHODS: We developed a sensitive immuno-PCR assay for the detection of TNFalpha in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFalpha sandwiched by antibodies was detected by amplification of the DNA label using PCR. RESULTS: The limit of detection of the assay was 0. 001 ng/L, an approximately 5 x 10(4)-fold improvement compared with a conventional ELISA. The mean serum TNFalpha concentration (+/- SD) in healthy donors was 0.021 +/- 0.044 ng/L in men (n = 29) and 0.033 +/- 0.065 ng/L in women (n = 25). CONCLUSION: This method may be useful for analyzing the significance of TNFalpha concentration in various diseases.

Heat-shock protein-73 protects against small intestinal warm ischemia-reperfusion injury in the rat.

BACKGROUND: The protective effects of heat-shock protein (hsp) in rat small intestinal warm ischemia-reperfusion (I/R) injury are poorly understood. METHODS: Hsp-73 expression was induced in rat small intestine with use of sodium arsenite injected (6 mg/kg) through a catheter cannulated into the left common carotid artery 24 hours before ischemia (group 1). In the control group an equal volume of phosphate-buffered saline solution was injected (group 2). To block the induction of hsp-73 expression, sodium arsenate and quercetin (5 mg/kg) were injected (group 3). RESULTS: The mean peak plasma levels of tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant after reperfusion were lower in group 1 than in group 2. The tissue myeloperoxidase activity after reperfusion was lower in group 1 than in group 2. The mean peak plasma level of interleukin-10 after reperfusion was higher in group 1 than in group 2. The induction of hsp-73 expression reduced the synthesis of nitric oxide and the magnitude of the small intestinal warm I/R injury. The results in group 3 were similar to those in group 2. CONCLUSION: Hsp-73 protects against small intestinal warm I/R injury by inhibiting the synthesis of inflammatory cytokines and the activation of neutrophils and by accelerating the synthesis of anti-inflammatory cytokines.

Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system.

We established the validity of a quantitative reverse transcription (RT)-PCR assay for the RNA component of human telomerase (hTR), using the TaqMan fluorogenic detection system. Using this assay, we quantified hTR expression in two human pancreatic cancer cell lines, ASPC-1 and MIAPaCa-2. Our results indicated that hTR expression in MIAPaCa-2 was 1.99-fold higher than that in ASPC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the amount of hTR in clinical specimens.

The plasma FK506-binding protein 12 level is related to acute cellular rejection in small bowel transplantation.

Since FK506 binding protein (FKBP12) inhibits dose-dependently the immunosuppressive activity of FK506 in vitro, plasma FKBP12 levels were measured after rat small bowel transplantation (SBTx). The mean plasma FKBP12 level in untreated recipients increased significantly at the onset of acute cellular rejection (ACR) compared to that in FK506-treated recipients without rejection at the same time after SBTx (P < 0.05). In both groups, however, the mean plasma FKBP12 level did not increase at 1 day after SBTx. These results suggest that plasma FKBP levels may be affected by ACR, but not by ischemia-reperfusion injury. Therefore, the plasma FKBP12 level should be considered as one of the parameters related to the immunosuppressive activity of FK506 in SBTx.

[The significance of telomerase activity in patients with gastric cancer].

The activation of telomerase is one of step in carcinogenesis. Therefore, it indicates that the detection of telomerase activity in tissues is useful for cancer diagnosis. TRAP assay developed by Kim et al. is sensitive enough to detect telomerase activity from a telomerase expressing cell. Using TRAP assay kit provided by Oncor Inc., we estimated quantitatively the telomerase activity from benign (atrophic gastritis), premalignant (intestinal metaplasia), and malignant tissues in the stomach. Telomerase activity in gastric cancer tissues was significantly higher than that in tissues which are characterized histologically as intestinal metaplasia or atrophic gastritis. In addition, to exclude interference with TRAP assay by telomerase or PCR inhibitor when telomerase activity was not observed in cancer tissues, the use of internal control (ITAS or TSNT) and dilution of samples should be performed.

Nonradioisotopic telomeric repeat amplification protocol (TRAP) using digoxigenin labeled probe.

We have attempted to detect telomerase activity by Southern blot analysis using a digoxigenin labeled probe (5'-(CCCTTA)6CCCTAA-3') Telomerase activity was detected in all cancer cell lines that were tested, and:the lower limit for detection was 10(3) cells. Although telomerase activity was detected in colon cancer tissues, it was not found in adenoma or normal tissues. Telomerase activity was completely abrogated by heat and RNase treatment of the CHAPS extract. Our results demonstrate that this nonradioisotopic assay is safe and reliable for detecting telomerase activity in cancer cell lines and biopsy samples.