Rapid, quantitative nonisotopic assay for telomerase activity in human tumors

Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 +/- 28.9, 29.2 +/- 26.7, and 35.3 +/- 15.9 ng DNA/microg protein, respectively, mean +/- SD), whereas gastric and endometrial cancers had a lower activity (17.2 +/- 8.8 and 13.5 +/- 7.9 ng DNA/microg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 +/- 12.9 and 2.8 +/- 2.1 ng DNA/microg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.     

Evaluation of an enzyme linked immunosorbent assay (ELISA) for determination of insulin in dogs

Between 1970 and 1983, 345 patients with ovarian cancer clinical stage I, II, and III were irradiated postoperatively. Five-year NED survival was achieved in 41.7% of patients. The most important prognostic factors were histological grade and clinical stage of cancer. Postoperative external beam radiotherapy appeared to be highly efficient for the patients with microscopic residual disease, giving 70% 5-year survival, and moderately efficient for patients with small, i.e. < or = 3 cm in diameter residual disease, giving 40% 5-year survival. The optimal technique of irradiation appeared to be the irradiation given to the entire abdominal cavity with additional irradiation coned down to the pelvis. External beam radiotherapy was ineffective in patients with gross residual disease, i.e. > 3 cm in diameter, and useless as palliative treatment given to patients with inoperable cancer of the ovary.     

Enzyme linked immunosorbent assay for detecting benzodiazepines in urine

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.     

Telomerase activity and metastasis: expansion of cells having higher telomerase activity within culture lines and tumor tissues

Tumor cells with metastatic potential may have a high telomerase activity that augments telomeric DNA repeats, allowing the cells to escape from the inhibition of cell proliferation due to shortened telomeres. We examined the expression level of telomerase activity using the telomeric repeat amplification protocol among a series of cell lines obtained by repeated transplantation of a mouse fibrosarcoma. The lines could be grouped into three; one has no metastatic potential, and the other two show metastatic abilities after intravenous or subcutaneous injection. Comparison of their telomerase activity indicated that more malignant lines had higher activity. A similar relation was seen in metastatic nodules formed through clonal expansion from the heterogeneous population of inoculated cells; clonality was monitored in terms of variable patterns of subtelomeric repeats. The results suggest that a high level of telomerase activity may not be requisite for metastasis, but may confer a propensity to dominate in a tumor tissue.     

Comparison of human telomerase RNA and telomerase activity in urine for diagnosis of bladder cancer

Currently the demand for transplant organs far outstrips the supply in the UK. This problem is even more severe for the Asian population, who have been shown to be disproportionately over-represented on transplant waiting lists in some regions of the UK. Several commentators have suggested that religious and cultural traditions may be the major determinant preventing Asians from donating organs. An exploratory qualitative study was undertaken with the aim of examining the influence of religious beliefs, amongst other things, on the extent and direction of public attitudes towards organ donation in a cross-section of the Asian population in Luton. This study indicates that, in the population studied, culture and religion play a much less prohibitive part in determining the level of organ donation than previously suggested. However, there is a desire to be aware of the religious stances so that people can make a more informed decision. The emphasis should clearly been a reconsideration of the presently inadequate approaches to organ procurement and on devising and supplementing these with more appropriate ones. An example of the failure to inform effectively the relevant populations about important developments is that only two of the 32 Muslims in the survey had heard of the 'fatwa' by the Muslim Legislative Council permitting organ donation.     

Expression of telomerase activity in human chorion

Telomerase activation is required for cellular immortalization and is found in most malignant tumors. Normal somatic cells are generally telomerase-negative, except for stem cells in renewing tissues. During pregnancy, human trophoblast continues to proliferate and acts as proliferating stem cells for the development of chorion and the formation of placenta. In the present study, a total of 105 chorions from placentas at various weeks of gestation were examined for telomerase activity using the telomeric repeat amplification protocol (TRAP) assay. Twenty-five of 33 (76%) normal early chorions at 5 to 9 weeks gestation were telomerase-positive. Chorions from early spontaneous abortions also exhibited telomerase activity but at a low level. In contrast, only 2 (4%) late chorions at 34 to 41 weeks gestation expressed telomerase activity. Significant telomerase activity was observed in trophoblast cell fractions of chorion, demonstrating trophoblast to be the source of the activity. Expression of human telomerase catalytic subunit (hTRT) was observed in early chorions, but not in late placenta, and there was a close correlation between telomerase activity and hTRT expression. In contrast, expression of human telomerase RNA component (hTR) was observed in both early and late chorions and was not liked to telomerase activity. These findings suggest that telomerase activity in chorion is critically regulated over the course of gestation, associated with hTRT expression. The findings of the present study also appear to support the emerging concept that normal somatic cells with stem cell-like characteristics can express telomerase activity.     

Detection of telomerase activity in oral lesions

We present a rare complication of pericardiectomy and the effective management thereof. A 67-year-old female with dyspnea and upper abdominal pain was received at our department upon referral. Chest roentgenogram and cardiac catheterization preceded a diagnosis of constrictive pericarditis. Pericardiectomy was performed subordinate to median sternotomy and left anterolateral thoracotomy. Extubation was carried out on day 2 but reintubation was necessitated on the same day as a result of progressive dyspnea. Chest roentgenogram revealed an atelectasis of the left lung. Fiberoptic bronchoscopy showed left main bronchial stenosis resulting from a pulsating external structure. A postoperative computed tomogram substantiated the stenotic left main bronchus between the dilated left pulmonary artery and the thoracic descending aorta. An expandable metallic stent for the treatment of this complication was selected over other invasive procedures. Two years of follow-up reveal no complications. Accordingly, an expandable metallic stent has demonstrated its effectiveness not only on bronchial stenosis due to malignancy or tuberculosis but on benign cases such as this as well.     

Regulation of telomerase activity in immortal cell lines

Telomerase is a ribonucleoprotein whose activity has been detected in germ line cells, immortal cells, and most cancer cells. Except in stem cells, which have a low level of telomerase activity, its activity is absent from normal somatic tissues. Understanding the regulation of telomerase activity is critical for the development of potential tools for the diagnosis and treatment of cancer. Using the telomeric repeat amplification protocol, we found that immortal, telomerase-positive, pseudodiploid human cells (HT1080 and HL60 cells) sorted by flow repressed in quiescent cells. This was true whether quiescence was induced by contact inhibition (NIH 3T3 mouse cells), growth factor removal (bromodeoxyuridine-blocked mouse myoblasts), reexpression of cellular senescence (the reversibly immortalized IDH4 cells), or irreversible cell differentiation (HL60 promyelocytic leukemia cells and C2C12 mouse myoblasts). Taken together, these results indicate that telomerase is active throughout the cell in dividing, immortal cells but that its activity is repressed in cells that exit the cell cycle. This suggests that quiescent stem cells that have the potential to express telomerase may remain unaffected by potential antitelomerase cancer therapies.     

Telomerase activity in normal and neoplastic breast

Telomerase activity is detected in the majority of human tumors and provides a mechanism to escape from proliferative limitations due to telomere loss. Similar to other studies, telomerase activity was detected with a modified telomeric repeat amplification protocol in the majority (76%) of 49 human breast cancer specimens, including most (75%) ductal carcinoma in situ specimens. There were no correlations between telomerase activity and tumor stage or estrogen/progesterone receptor status. In four of seven invasive tumors, telomerase expression seemed to be heterogeneous because not all microdissected regions were telomerase positive. Low levels of telomerase activity were also detected in a minority (17%) of breast specimens from patients without evidence of cancer. These findings suggest that telomerase activation can occur early in breast cancer progression and may be periodically down-regulated during subsequent progression.     

Evaluation of an enzyme immunosorbent assay for the diagnosis of Argentine haemorrhagic fever

To elaborate a set of serological tests for the diagnosis of Argentine haemorrhagic fever (AHF), an enzyme-linked immunosorbent assay (ELISA) for detection of specific anti-Junin virus (JV) IgG is described, and its performance is compared with that of the plaque reduction neutralization test (PRNT). The reproducibility, sensitivity, specificity, and confidence limits for positive and negative results for ELISA were statistically analysed. The value of 800 was demonstrated as the lowest positive titer. Titers > or = 800 varied within one (two-fold) dilution in 95.6% of the tests, while the sensitivity and specificity were 99.2% and 98.8%, respectively. The assay yielded 1% of false positives and 0.05% of false negatives. A comparison of ELISA to PRNT in detecting the seroconversion for JV was studied by the chi square test (comparison of proportions in paired samples) and the K parameter for agreement proportion. Comparison of ELISA to PRNT showed no significant difference in the proportions of positive and negative results of these assays (P < 0.01), demonstrating an equivalent performance (K = 0.98) in the diagnosis of AHF. In addition, the simplicity and safety of the procedures involved make this ELISA the most suitable test to detect natural human JV infections.     

Evaluation of somatostatin-receptor scintigraphy for detecting neuroendocrine tumors

BACKGROUND: Somatostatin-receptor scintigraphy (SRS) has gained attention as an imaging modality for neuroendocrine tumors (NETs). The purpose of this study was to present one of the first American series evaluating the ability of SRS to detect local and distant disease caused by NETs. METHODS: Medical records were reviewed from 35 patients who underwent a total of 38 studies using 111In-pentetreotide between 1993 and 1995. Twenty-two patients had islet cell tumors, seven had carcinoid tumors, and six had other NETs. RESULTS: The overall sensitivity of SRS was 74% for detecting local disease (primary tumor +/- regional lymph node metastases) in all NETs, excluding insulinoma, 75% in gastrinoma, 0% in insulinoma, 78% in other islet cell tumors, and 50% in carcinoids. For detecting distant disease, the overall sensitivity of SRS was 67% for all NETs, excluding insulinoma, 100% for gastrinoma, 50% for other islet cell tumors, and 80% for carcinoids. Specificity and positive predictive value were 100% for all tumors. Negative predictive value ranged from 33% to 100%. CONCLUSIONS: A positive SRS study strongly predicts the presence of tumor (100% positive predictive value in our study). However, unlike the European reports of very high sensitivity (80% to 88%), we found that SRS had a lower sensitivity (67% for all NETs excluding insulinoma and 71% for noninsulinoma gastroenteropancreatic NETs). Thus negative SRS in patients with NETs must be viewed cautiously, because the false-negative rate is high, and this limits the use of this method in the most difficult patients.     

Radioisotopic assay of vitamin B12 in tissues

The present study delineates the application of the radioisotopic competitive-inhibition assay for the measurement of vitamin B12 in tissues. The extraction of endogenous B12 from tissues was shown to be simple and complete. Proportional dilution studies suggest that tissue factors do not interfere with the assay. Although some variability exists when multiple areas of an organ are sampled, the differences between B12 levels in tissues obtained from normal individuals and B12-deprived individuals are so wide that individual intra-organ variability is trivial, As this tissue B12 assay is similar to the widely utilized radioisotopic assays for serum B12, it is applicable for routine use.     

Clinical implications of telomerase activity levels in acute leukemia

In the present study, we used the telomeric repeat amplification protocol assay, an internal telomerase assay standard, and an automatic DNA sequencer to detect and quantitate telomerase activity in blood samples obtained from normal and acute leukemia patients. Telomerase activity was analyzed in 78 acute leukemia patients and ranged from 0.65 to 147 relative to the internal standard. Compared to the age-matched normal levels of telomerase activity in the peripheral blood cells, we determined that 45 (81.8%) of 55 acute myeloid leukemia (AML) and 16 (69.6%) of 23 acute lymphoid leukemia patients had elevated telomerase activity. There was no relationship between peak telomere length and telomerase activity in both acute lymphoid leukemia and AML patients. In AML, the level of telomerase activity was associated with French-American-British subtypes and cytogenetics, and patients with elevated telomerase activity had high leukocyte counts and more frequent extramedullary involvement during the disease. Among 78 patients, 5 had high levels of telomerase activities similar to immortalized leukemia cell lines; these 5 patients had a very poor prognosis (P < 0.05). The levels of telomerase activity significantly decreased in patients in complete remission. Most of the patients in complete remission showed a normal level of telomerase activity; however, two of them had low to moderate telomerase activity, and they relapsed shortly after entering complete remission. In relapsed patients, there is a general trend for increased telomerase levels, and 2 of the 13 patients retained high telomerase activity, whereas the other 11 had normal to moderate telomerase activity. These results suggest that telomerase activity may be a useful additional method for monitoring the disease condition in acute leukemia patients.