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J W Shay

Publications and source records attributed to J W Shay.

At least 109 records · Page 6Linked to original sources
PubMed1997

Telomerase activity in neuroblastoma: is it a prognostic indicator of clinical behaviour?

Neuroblastomas show remarkable biological heterogeneity, resulting in favourable prognosis or unfavourable prognosis due to aggressive growth despite multimodal therapy. Recently, we proposed that aggressive tumours express telomerase at a high level while the favourable tumours lack or have low telomerase expression. To evaluate the correlation between telomerase activity and other biological characteristics reported as prognostic markers (MYCN gene amplification, loss of heterogeneity (LOH) in the short arm of chromosome 1, trk-A expression, Ha-ras p21 expression, and DNA ploidy), we investigated these biological features in 105 untreated neuroblastomas. In these cases, 23 showed high telomerase activity, 78 showed low activity, and telomerase activity was undetectable in 4 cases. Most tumours with genetic alterations (MYCN amplification or 1p32 LOH) showed high telomerase activity. Most tumours with low or undetectable activity were aneuploid, and showed trk-A and Ha-ras expression. Three of the four tumours with undetectable telomerase activity regressed. In 2 of the tumours with low telomerase activity, the residual tumours maturated and showed repression of telomerase activity. Thus, the level of telomerase activity correlated with other genetic alterations and/or gene expression and may be a useful prognostic indicator in neuroblastoma.

Age Factors↗
PubMed1997

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Amino Acid Sequence↗
PubMed1997

Telomerase activity and cytogenetic changes in chronic myeloid leukemia with disease progression.

Progressive telomere shortening is thought to be important in the regulation of cellular senescence and that the upregulation or reactivation of telomerase activity may be a critical if not rate limiting step in the development of neoplastic cells. To obtain information about telomeres and telomerase activity in hematopoietic neoplasia at various disease stages, we evaluated 54 samples obtained from 41 patients with chronic myeloid leukemia (CML) using a combination of fluorescent-telomeric repeat amplification protocol and an internal telomerase assay standard. The terminal restriction fragment (TRF) lengths in the blast phase was reduced compared to that in the chronic phase (4.53 +/- 0.72 kb vs 6.13 +/- 1.68 kb; P = 0.0005). All samples obtained from CML in the chronic phase (n = 33) had detectable telomerase activity above background, regardless of age. In the blast phase (n = 21), a significant increase of telomerase activity was detected compared to that in the chronic phase (33.84 +/- 37.86% vs 6.08 +/- 3.21; P = 0.016). Among patients in the blastic phase, 50% of patients had moderate to high telomerase activity (>10 relative value), and the remaining patients had telomerase activity higher than that in the normal peripheral blood cells. No significant differences in hematologic findings, duration of chronic phase or blast phase, and telomere length in the blastic phase were noted between these two groups separated by telomerase activity. CML patients with moderate to high telomerase activity had a high frequency of additional cytogenetic changes (P = 0.01).

Adolescent↗
PubMed1997

Expression of telomerase RNA component correlates with the MIB-1 proliferation index in ependymomas.

Although there is general agreement that certain morphologic subtypes of ependymoma are benign, the biologic behavior of other ependymal neoplasms is poorly understood and not clearly related to conventional histopathologic criteria. The absence of universally accepted standards has prompted the search for more objective biologic markers. Telomerase is an RNA-containing enzyme associated with immortality in proliferating stem cells and many tumors. We investigated the proliferative activity of 26 ependymomas as determined by MIB-1 immunolabeling and compared the results with the in situ expression of human telomerase RNA (hTR) and WHO tumor grade. The study included 9 WHO grade I ependymomas (6 subependymomas and 3 myxopapillary ependymomas), 13 WHO grade II ependymomas, and 4 anaplastic (WHO grade III) ependymomas. The proliferation index (PI) and telomerase RNA expression were significantly increased in grade III ependymomas (p < 0.0001 for PI and p = 0.0015 for hTR). In these tumors, the PI and hTR expression were highly correlated (p = 0.0001). Of note, a single case designated grade II showed both increased proliferative activity and the highest hTR expression detected in this series of ependymal neoplasms. Our results suggest that the PI and hTR expression may be important biologic markers, independent of other histopathologic criteria of tumor grade. Future studies examining the correlation of MIB-1 cell kinetics and hTR expression with clinical parameters in selected ependymoma subtypes are needed to determine the prognostic relevance of these markers.

Adolescent↗
PubMed1997

Human telomerase RNA expression and MIB-1 (Ki-67) proliferation index distinguish hemangioblastomas from metastatic renal cell carcinomas.

Hemangioblastomas are low-grade, capillary rich neoplasms of the cerebellum and spinal cord that can occur sporadically or in the setting of Von Hippel-Lindau syndrome. The present study analyzed the utility of proliferation potential in differentiating hemangioblastoma from RCC metastatic to the central nervous system using a MIB-1 (Ki-67) labeling index and assessment of expression of the RNA component of telomerase. Immunohistochemical analysis for epithelial membrane antigen (EMA) and MIB-1 was performed on paraffin-embedded sections of 27 hemangioblastomas and 5 RCC metastatic to the central nervous system. All but one hemangioblastoma demonstrated low or negative MIB-1 immunoreactivity, while 4 of 5 RCC metastases had moderate or high labeling indices. Telomerase RNA expression was assessed in 10 hemangioblastomas and in all 5 metastatic RCC by in Situ hybridization. All 10 hemangioblastomas demonstrated a lack of expression of telomerase RNA, while all 5 metastatic RCCs showed moderate to strong expression. Our results suggest that the MIB-1 labeling index is useful in differentiating hemangioblastoma from metastatic RCC and assessment of telomerase expression can also provide novel information on the difference in growth potential of these tumors.

Adolescent↗
PubMed1997

Telomerase activity concentrates in the mitotically active segments of human hair follicles.

Telomerase is a ribonucleoprotein enzyme capable of adding hexanucleotide repeats onto the ends of linear chromosomal DNA. Whereas normal somatic cells with a limited replicative capacity fail to express telomerase activity, most immortal eukaryotic cells do. Cells of renewal tissues (e.g., skin, intestine, blood) require an extensive proliferative capacity. Some cells in such renewal tissues also express telomerase activity, most likely to prevent rapid erosion of their telomeres during cell proliferation. In this study, we measured the levels of telomerase activity in dissected compartments of the human hair follicle: hair shaft, gland-containing fragment, upper intermediate fragment (where it is thought undifferentiated stem cells reside), lower intermediate fragment, and in the bulb-containing fragment (an area with high mitotic activity containing a more differentiated pool of keratinocytes). In anagen follicles, high levels of telomerase activity were found almost exclusively in the bulb-containing fragment of the follicles, with low levels of telomerase in the bulge area (intermediate fragments) and gland-containing fragment. In comparison, catagen follicles had low levels of telomerase activity in the bulb-containing fragments as well as in other compartments. Such observations indicate that, in anagen hair follicles, the fragments containing cells actively dividing (e.g., transient amplifying cells) express telomerase activity, whereas fragments containing cells with low mitotic activity, for example, quiescent stem cells, express low levels of telomerase activity.

Adult↗
PubMed1997

Telomerase in the early detection of cancer.

Although there is justifiable optimism regarding telomerase activity and early detection of cancer, it is important to point out that there is much that remains to be understood and additional validation studies will be required before knowledge of telomerase activity will be useful in decisions regarding patient management. A key question is whether we will be able to distinguish those cancers that are going to progress from those cancers that are not by detecting telomerase activity. Molecular staging using markers such as telomerase activity in combination with other molecular markers may be particularly useful in this regard. Basic scientists and clinicians must work together if this strategy is to mature. Telomerase inhibitors in the treatment of cancer are awaited with great anticipation.

Biomarkers, Tumor↗
PubMed1997

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.

Frozen Sections↗
PubMed1997

Decline in telomerase activity as a measure of tumor cell killing by antineoplastic agents in vitro.

Telomerase activity is frequently associated with the malignant phenotype, and it can be considered an almost ubiquitous tumor marker. In this study, we evaluated telomerase activity in telomerase-positive human tumor cell lines exposed in vitro to antineoplastic agents. The results show that drug-induced cell killing of tumor cells is associated with a decline in detectable telomerase activity. The decrease of telomerase activity levels paralleled cell growth impairment evaluated by cell count or by measurement of cell ability to convert tetrazolium salt to colored formazan [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide assay]. The observed telomerase activity remaining after treatment with antineoplastic agents is most likely to reflect activity from the remaining viable cells. When tumor cell lines resistant to the chemotherapeutic agents temozolomide or doxorubicin were treated with these compounds, no decline of telomerase activity or cell growth was observed. The results of the present study indicate that resistance of neoplastic cells to chemotherapeutic agents can be monitored by following telomerase activity. Moreover, the test can be performed with a limited number of neoplastic cells, such as those frequently obtained from tumor biopsies. These findings provide a rationale for developing new in vitro chemosensitivity assays, and detection of telomerase activity may be a novel marker of chemotherapy failure.

Antineoplastic Agents↗
PubMed1997

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.

Adolescent↗
PubMed1996

Chromosome end-to-end associations and telomerase activity during cancer progression in human cells after treatment with alpha-particles simulating radon progeny.

Chromosome end-to-end associations seen at metaphase involve telomeres and are commonly observed in cells derived from individuals with ataxia telangiectasia and most types of human tumors. The associations may arise because of short telomeres and/or alterations of chromatin structure. There is a growing consensus that telomere length is stabilized by the activity of telomerase in immortal cells; however, it is not clear why some immortal cells display chromosome end-to-end associations. In the present study we evaluated chromosome end-to-end associations, telomere length and telomerase activity with the tumorigenic status of human bronchial epithelial cells immortalized with human papillomavirus. Oncogenic transformation was initiated using radon simulated alpha-particles and cells evaluated as primary, secondary and metastatic transformants. The fewest chromosome end associations and lowest telomerase activity were observed in the parental immortalized cells. However, increased levels of telomerase activity were detected in alpha-particle survivors while robust telomerase activity was seen in the tumorigenic cell lines. The tumorigenic cells that were telomerase positive and had the highest frequency of cells with chromosome end-to-end associations were also metastatic. No correlation was found between telomere length and the different stages of carcinogenicity.

Ataxia Telangiectasia↗
PubMed1996

Telomerase activity in human renal cell carcinoma.

Telomeres have a vital role in maintaining chromosome stability and are essential for long term viability. Since the very ends of linear chromosomes cannot replicate, telomeres shorten in normal somatic cells eventually resulting in growth inhibition. However, most immortal cell lines maintain stable telomeres indicating that mechanisms exist to compensate for the end replication problem. Telomerase activity, leading to synthesis of telomeric DNA repeats, has been proposed to be an important step in the immortalization process of tumor cells. In the present study, 56 renal cell carcinomas were tested for telomerase activity using the sensitive TRAP (telomeric repeat amplification protocol). Forty of the analysed tumors (71%) were positive for telomerase activity, whereas none of the 56 corresponding normal kidney samples showed telomerase activity. All telomerase negative tumors had a reduction in mean telomere restriction fragment (TRF) length and a decrease in total telomere repeat hybridization signal, though cases were observed with an increase in peak TRF lengths. No obvious association between the presence of telomerase activity and clinicopathological parameters (histopathologic grade, DNA-ploidy, stage and clinical outcome) was found. The high frequency of detection of telomerase activity in the renal cell carcinomas indicates that this enzyme is likely to be an important factor involved in the evolution of this tumor type.

Base Sequence↗
PubMed1996

Experimental elongation of telomeres extends the lifespan of immortal x normal cell hybrids.

Hybrids between immortal cells that express telomerase and normal cells that lack telomerase have a limited lifespan. We demonstrate that telomerase is repressed in such hybrids. Treatment of immortal human cell lines with certain oligonucleotides resulted in telomere elongation. We took advantage of this observation to test the hypothesis that elongation of telomeres would extend the lifespan of cells in culture. An immortal human cell line was treated with an oligonucleotide to lengthen its telomeres and then was fused with mortal cells. The lifespan of these hybrid cells was longer than that of the hybrids in which telomeres had not been elongated. These observations provide the first direct evidence supporting the hypothesis that telomere length determines proliferative capacity of human cells.

Base Sequence↗
PubMed1996

Immortalization of human mammary epithelial cells transfected with mutant p53 (273his).

Normal human breast epithelial cells were transfected with expression vectors containing the p53 gene mutated at either codon 143, 175, 248 or 273, or by infection with a recombinant retroviral vector containing the p53 gene mutated at codons 143, 175, 248, or 273. The breast epithelial cells were monitored for extension of in vitro lifespan and immortalization. Expression of some, but not all, p53 mutants resulted in an extension of in vitro lifespan. Experiments with the p53 temperature sensitive mutant 143ala revealed that at 32 degrees C, the nonpermissive temperature, the growth of breast epithelial cells was inhibited. At 37 degrees C, the mutant conformation, there was increased proliferation of cells, resulting in extension of in vitro lifespan. Breast epithelial cells expressing p53 mutant 273his maintained DNA binding and transcriptional activities and one clone immortalized after a period of growth arrest (crisis). The progression of this immortalization event was characterized by the reactivation of telomerase using the telomeric repeat amplification protocol (TRAP), and terminal restriction fragment analysis (TRF). This is the first reported immortalization of human mammary epithelial cells transfected with a mutant 53.

Amino Acid Sequence↗
PubMed1996

Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues.

Telomerase activity has been detected in many human immortal cells lines and in tumor tissues, whereas it is generally absent from primary cell strains and from many tumor adjacent tissue samples. With the recently cloned human telomerase RNA (hTR), we used Northern analysis to follow the levels of hTR in primary, precrisis, and immortalized cells. It was surprising that the amount of hTR was high in cell strains that lacked telomerase activity, and the levels did not parallel the increases in telomerase activity, which accompanies immortalization. In addition, although the hTR levels were somewhat higher in tumor samples compared to nontumor tissues, the level of hTR in a variety of different human tumors did not predict the level of telomerase activity in the tumor. Thus, whereas hTR was detected in all samples that have telomerase activity, the presence of the RNA was not a good predictor of the presence or amount of telomerase activity.

B-Lymphocytes↗
PubMed1996

Telomerase activity in human breast tumors.

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is not detected in normal somatic cells; thus, with each cell division, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode. The current model gaining support is that telomerase activity in germline and immortal cells maintains telomere length and thus compensates for the "end-replication problem." PURPOSE: Our objective was to determine when telomerase activity is reactivated in the progression to malignant breast cancer and if knowledge of telomerase activity may be an indicator for the diagnosis and potential treatment of breast cancer. METHODS: Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 140 breast cancer specimens (from 140 patients), four phyllodes tumors (from four patients), 38 noncancerous lesions (20 fibroadenomas, 17 fibrocystic diseases, one gynecomastia; from 38 patients), and 55 adjacent noncancerous mammary tissues (from 55 of the 140 breast cancer patients). In addition, 33 fine-needle-aspirated breast samples (from 33 patients) were analyzed. RESULTS: Among surgically resected samples, telomerase activity was detected in 130 (93%) of 140 breast cancers. Telomerase activity was detected in 68% of stage I primary breast cancers, in 73% of cancers smaller than 20 mm, and in 81% of axillary lymph node-negative cancers. Moreover, the activity was detected in more than 95% of advanced stage tumors but in only two (4%) of 55 adjacent noncancerous tissues. While telomerase activity was not detected in any of 17 specimens of fibrocystic disease, surprisingly low levels of telomerase activity were detected in nine (45%) of 20 fibroadenomas. Among samples obtained by fine-needle aspiration, 14 (100%) of 14 patients whose fine-needle-aspirated specimen contained telomerase activity and who subsequently underwent surgery were confirmed to have breast cancer. Multivariate analysis of 125 specimens from patients for whom data were available on age at surgery, stage of disease, tumor size, lymph node status tumor histology, and menopausal status indicated that stage classification exhibited the strongest association with telomerase activity (for stage I versus stages II-IV: odds ratio = 1.0 versus 73.4; 95% confidence interval = 2.0-959.0; P = .02). CONCLUSION: Telomerase activity was detected in more than 95% of advanced stage breast cancers. It was absent in 19%-32% of less advanced cancers. Since a determination of any association between telomerase activity and patient survival is not possible at the present time, it remains to be determined whether lack of telomerase activity predicts for favorable outcome.

Adult↗
PubMed1996

Telomerase activity: a prevalent marker of malignant human prostate tissue.

We urgently need biochemical markers to detect the malignant nature and pathological states of the human prostate. We report that telomerase activity is associated with prostate cancer but absent in the benign disease and normal gland. Telomerase is, therefore, a potential diagnostic marker for prostate cancer. Twenty-five human prostates resected at the time of radical prostatectomy were dissected to obtain matched adjacent areas of normal, central zone benign prostatic hyperplasia (BPH), and pathologically confirmed cancer tissue. These matched tissue samples were assayed for telomerase activity using a sensitive PCR technique. None of the normal tissues exhibited telomerase activity. In contrast, 21 of the 25 (84%) cancers were strongly positive. At the time of prostatectomy, four lymph nodes were positive for metastases and all were strongly positive for telomerase activity. In adjacent BPH tissues taken from the cancerous prostates, only 3 of the 25 tissues (12%) were weakly positive. Telomerase activity was not detected in ten BPH samples recovered from patients who underwent open surgery solely for BPH. All five available cell lines of human prostate cancer (DU145, LNCaP, PC3, PPC1, and TSU) were strongly positive. Short telomere lengths have been observed in several human cancers. We also measured the telomere lengths in 27 matched samples of normal, BPH, and cancer tissue taken from nine radical prostatectomies. The telomeres from cancer tissue were significantly and consistently shorter than either the adjacent normal or adjacent BPH tissues. Our results indicate that telomerase activity, as well as telomere lengths, may be markers for distinguishing prostate cancer from normal and benign prostate tissues.

Aged↗